Methods for the production of plants resistant to HPPD herbicides

ABSTRACT

Methods for making transgenic plants that are resistant to HPPD herbicides are presented. Polynucleotides other than those from  Pseudomonas fluorescens  that encode resistant HPPD enzymes are enclosed for use in the process of making transgenic plants that are tolerant to HPPD-inhibiting herbicides.

FIELD OF THE INVENTION

The present invention relates to recombinant DNA technology, and inparticular to the production of (i) transgenic plants which exhibitsubstantial resistance or substantial tolerance to herbicides whencompared with non transgenic like plants; and (ii) transgenic plantswhich contain relatively elevated levels of lipid soluble anti-oxidants,likewise when compared with non-transgenic such plants. The inventionalso relates, inter alia, to the nucleotide sequences (and expressionproducts thereof) when used in the production of, or when produced by,the said transgenic plants.

BACKGROUND OF THE INVENTION

Plants which are substantially “tolerant” to a herbicide when they aresubjected to it provide a dose/response curve which is shifted to theright when compared with that provided by similarly subjected nontolerant like plants. Such dose/response curves have “dose” plotted onthe x-axis and “percentage kill”, “herbicidal effect” etc. plotted onthe y-axis. Tolerant plants will typically require at least twice asmuch herbicide as non tolerant like plants in order to produce a givenherbicidal effect. Plants which are substantially “resistant” to theherbicide exhibit few, if any, necrotic, lytic, chlorotic or otherlesions when subjected to the herbicide at concentrations and rateswhich are typically employed by the agricultural community to kill weedsin the field.

Within the context of the present invention the terms hydroxy phenylpyruvate (or pyruvic acid) dioxygenase (HPPD), 4-hydroxy phenyl pyruvate(or pyruvic acid) dioxygenase (4-HPPD) and p-hydroxy phenyl pyruvate (orpyruvic acid) dioxygenase (p-OHPP) are synonymous.

Methods for providing plants which are tolerant to HPPD herbicides whichcomprise transformation of plant material with polynucleotidescomprising regions which encode HPPD enzymes are known. However what hasnot hitherto been generally recognized is that different HPPD enzymesprovide different levels of tolerance to different HPPD-inhibitorherbicides. While a given HPPD enzyme may provide a useful level oftolerance to some HPPD-inhibitor herbicides it may be quite inadequateto provide commercial levels of tolerance to a different, more desirableHPPD-inhibitor herbicide which, for example, may control a differentspectrum of weeds, be cheaper to make or offer environmental benefits.As well as particular HPPD enzymes and the polynucleotides which encodethem the current invention also provides a means of selecting HPPDenzymes suitable for providing commercially useful levels of resistanceto particular HPPD-inhibitor herbicide chemistries.

In order to provide for plants with tolerance to commercially usefulapplication rates of a desired HPPD herbicide it would be an advantageto use polynucleotides which encode HPPD enzymes having reducedsusceptibility to inhibition by the desired HPPD herbicide or class ofHPPD herbicides. This characteristic of reduced susceptibility toinhibition by HPPD herbicides in vitro is also expressed herein as‘increased resistance’ or ‘inherent tolerance’.

Some mutant forms of a Pseudomonas sp. HPPD are claimed to exhibit suchincreased resistance on the basis of exhibiting an apparently decreasedrate of binding of inhibitor to the enzyme (i.e., on the basis ofmeasurements essentially corresponding to k_(on) in the equilibrium E+I← → EI, vide infra). However such mutant enzyme forms have reducedcatalytic activity and/or reduced stability which, potentially, rendersthem unsuitable for use especially in the warm climate crops,particularly corn and soybean for which HPPD-inhibitor herbicidesgenerally provide the most useful spectrum of weed control. It has nothitherto been known that various unmutated wild-type HPPD enzymes fromdifferent sources can equally exhibit useful and different inherentlevels of tolerance and that, furthermore, unmutated wild-type enzymesare preferable for use in transgenic plants because, in general, theyexhibit considerably better stability and activity (kcat/Km) than themutant derivatives.

Furthermore it has not hitherto been appreciated that the level ofinherent tolerance of these wild-type HPPD enzymes or indeed of mutatedHPPD enzymes can vary markedly according to the particular class andstructure of HPPD inhibitor. Neither has it been known that thesedifferences in tolerance have their basis not in differences in theparameter k_(on), addressed by previously used assay methods, butrather, in the parameters Kd, and k_(off). It has also not beenappreciated that these differences in inherent tolerance can be markedand useful even between HPPD enzymes having relatively similar aminoacid sequences as, for example, between sequence similar HPPD enzymesderived from different species of plants. In order to maintain thewidest range of options for herbicide modes of action useful for thecontrol of volunteer crops and to minimize any potential impact of geneflow to weeds it is desirable that the herbicide tolerance conferredupon transgenic plants be expressed preferentially toward only certaindesired subclasses of HPPD inhibitor herbicides. This is another benefitof being able to choose a particular HPPD enzyme most suited todelivering resistance to a particular set of HPPD herbicide types.

SUMMARY OF THE INVENTION

The present invention relates to nucleotide sequences, derivativenucleotide sequences, and the like, encoding amino acid sequences havingenzymatic activity such that the amino acid sequences are resistant toHPPD inhibitor herbicidal chemicals. The present invention also relatesto methods for making transgenic plants that are resistant to HPPDherbicides due to the expression of resistant HPPD enzymes. The presentinvention also relates to methods for screening for new resistant HPPDenzymes, as well as to methods for controlling weeds in crop fields byspraying HPPD chemicals over the top of transgenic crop plantsexpressing resistant HPPD enzymes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the rate of exchange of C14 mesotrione from wheat and fromArabidopsis HPPD.

FIG. 2 shows the rate of exchange of C14 mesotrione and of structure IIbound to Arabidopsis HPPD with excess 14C mesotrione.

FIG. 3 shows the progressive inhibition of wheat HPPD by structure II.

FIG. 4 shows the progressive inhibition of wheat HPPD by structure VI.

FIG. 5 shows the binding of C14 mesotrione to Arabidopsis HPPD.

DETAILED DESCRIPTION OF THE INVENTION

When the word “specific” is used in conjunction with the resistance of aparticular protein to a particular herbicide—or class of herbicide, theterm obviously does not exclude some degree of sensitivity-especially inthe case that high levels (non-commercial application rates) ofherbicidally are applied.

By “triketone herbicide” is meant a derivative of a cyclohexane 1,3dione or a bicyclo [3,2,1]octane-2-4dione.

By “syncarpic acid” is meant a derivative of a4,4,6,6-tetramethylcyclo-hexane 1,3,5-trione.

According to the present invention there is provided a triketoneinhibitor specific resistant—HPPD enzyme comprising an amino acidsequence QIKECQ (SEQ ID NO: 33) and a sequence F, (D/E), F, (M/L), W1,(P/A), P, W2, X, X, Y, Y (SEQ ID NO: 34) wherein W1 is either A or P andwhere (i) if W1 is A then W2 is P, A, Q or L, or (ii) if W1 is P then W2is P, A, Q or T, wherein X is any amino acid.

The present invention also provides a triketone inhibitor specificresistant HPPD enzyme comprising an amino acid sequence PPTPT (SEQ IDNO: 35) and a sequence F, (D/E), F, (M/L), W1, (P/A), P, W2, X, X, Y, Y(SEQ ID NO: 34) wherein W1 is either A or P and where (i) if W1 is Athen W2 is P, A, Q or L, or, if (ii) W1 is P then W2 is P, A, Q or T,and X is any amino acid.

In a preferred embodiment of the present inventive enzyme the enzymefurther comprises at least one of the following sequences: (i) (L/V), A,S, X, D, V, L (SEQ ID NO:36) (ii) (R/Q), A, R, (S/T), (P/A), M, G, G(SEQ ID NO:37) (iii) (K/D/E/N), Y, Y, (D/E), G, V, R, R (SEQ ID NO:38)(iv) Q, E, L, G, V, L (SEQ ID NO:39) (v) (H/Y), (H/N), G, G, (P/S), G, V(SEQ ID NO:40) (vi) E, K, D, E, (R/V/K/Q), G, (Q/R/E), E (SEQ ID NO:41)where X is any amino acid.

The present inventive HPPD-inhibitor resistant HPPD enzyme may be ableto -form a complex with an HPPD inhibitor of Structure I wherein thedissociation constant (Kd) of said complex, in water at pH 7.0 and at 25C, is within the range from 1.0 to 30 nM and/or the dissociation rateconstant of the complex, in water at pH 7.0 and at 25 C, is within therange of from 4×10⁻⁵ to 2×10⁻³ s⁻¹. STRUCTURE I.2-(Nitro-4-methanesulphonylbenzoyl)-cyclohexane-1,3-dione

The kcat/Km hydroxyphenylpyruvate value of the HPPD-inhibitor resistantHPPD enzyme may be in the range of from 0.8 to 5.0 s⁻¹ μM⁻¹ at pH 7.0,and 25° C.

The present invention also provides an HPPD inhibitor resistant HPPDenzyme excluding those derived from maize, wheat and barley,characterised in that in comparison with an Arabidopsis derived HPPDenzyme, the resistant enzyme exhibits at least a 2.5 and preferably afour fold increased resistance to herbicides selected from those ofFormula I and/or Formula 2 as compared to herbicides selected fromFormula 3 and/or Formula 4 as depicted below. Note that whereverstructures are drawn in a keto form that these structures can also existin an enolic form and that all of these and all other tautomeric formsare also included within the formula.

where Ar groups A, B. D and E are independently chosen from optionallysubstituted phenyl or optionally substituted heteroaryl. R₁ or R₂ orboth are H and both R₃ and R₄ are H and R₅ or R₆ or both are H. R₈ andR₉ are both H and R₁₀ or R₁₁l or both are H. R₁₂ or R₁₃ or both are Hand R₁₄ or R₁₅ or both are H. Aside from these constraints, R₁-R₁₇ areeach individually selected from the group consisting of H. —C₁-C₄ alkyl,C₃-C₆ cycloalkyl, halogen, OH, SH, CN, —NH₂, —NHCOR, —CONHR, —COR, —SR,SOR, —SO₂R, NHR—SO₂R, —CO₂R, —NO₂, CF₃, —SF₅, OR, and CO₂H where R=C₁-C₆alkyl oraryl optionally substituted with one or more substituentsselected from the group consisting of halo or C₁-C₄ alkoxy.

Optional substituents for the groups A, B, D and E include —C₁-C₄ alkyl,C₃-C₆ cycloalkyl, halogen, OH, SH, CN, —NH₂, —NHCOR, —CONHR, —COR, —SR,SOR, —SO₂R, NHR—SO₂R, —CO₂R, —NO₂, CF₃, —SF₅, OR, and CO₂H where R=C₁-C₆alkyl or aryl optionally substituted with one or more substituentsselected from the group consisting of halo or C₁-C₄ alkoxy.

In a preferred embodiment of the method Ar is substituted phenyl andR₁₋₃, R₅ and R₆ are each H and R₄ is not H. Alternatively, in a morepreferred embodiment, Ar may be substituted phenyl and R₁-R₆ are all H.The said phenyl may have H at all positions other than 2 and 4, whichare then preferably substituted at position 2 with NO₂ or Cl and atposition 4 with SO₂Me or Cl.

In a further preferred embodiment Ar is a substituted 3-pyridyl.Optionally the pyridyl N may be N-oxide. The said pyridyl may have H atall positions other than 2 and 6, which are then preferably substitutedat position 2 with R′ and at position 6 with CF₂H, CF₂Cl or CF₃ andwhere R′ is Me, isopropyl, n-propyl, CH₂OMe, CH₂OEt, CH₂CH₂OMe or CF₃.

Herbicidal HPPD inhibitors of Formula 1 include their agronomicallyacceptable salts. According to particular preferred embodiments (i)polynucleotides of the invention are selected to encode HPPD inhibitorresistant HPPD enzymes and (ii) plants are produced which aresubstantially tolerant to representative examples of herbicide Formula Isuch as

2-(2—Nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione and/or

2-(2-Chloro-4-methanesulphonylbenzoyl)-cyclohexane-1,3-dione, and/or

2-(Nitro-4-methanesulphonylbenzoyl)-cyclohexane-1,3-dione, the secondand third of which are known respectively as sulcotrione and mesotrione.

where group G is chosen from optionally substituted phenyl or optionallysubstituted heteroaryl. R₁-R₂ are each individually selected from thegroup consisting of H, —C₁-C₄ alkyl, C₃-C₆ cycloalkyl, halogen, OH, SH,CN, —NH₂, —NHCOR, —CONHR, —COR, —SR, SOR, —SO₂R, NHR—SO₂R, —CO₂R, —NO₂,CF₃, —SF₅, OR, and CO₂H where R=C₁-C₆ alkyl or aryl optionallysubstituted with one or more substituents selected from the groupconsisting of halo or C₁-C₄ alkoxy.

Preferably both R₁ and R₂ are H.

Optional substituents for the group G, include —C₁-C₄ alkyl, C₃-C₆cycloalkyl, halogen, OH, SH, CN, —NH₂, —NHCOR, —CONHR, —COR, —SR, SOR,—SO₂R, NHR—SO₂R, —CO₂R, —NO₂, CF₃, —SF₅, OR, and CO₂H where R=C₁-C₆alkyl or aryl optionally substituted with one or more substituentsselected from the group consisting of halo or C₁-C₄ alkoxy or C₁-C₄alkoxyalkoxy.

Herbicidal HPPD inhibitors of Formula 2 include their agronomicallyacceptable salts. In a preferred embodiment Ar is a substituted3-pyridyl and R₁ and R₂ are both H. Optionally the pyridyl N may beN-oxide. The said pyridyl may have H at all positions other than 2 and6, which are then preferably substituted at position 2 with R′ and atposition 6 with CF₂H, CF₂CI or CF₃ and where R′ is Me, isopropyl,n-propyl, CH₂OMe, CH₂OEt, CH₂CH₂OMe or CF₃. According to particularpreferred 1s embodiments (i) polynucleotides of the invention areselected to encode HPPD-inhibitor resistant HPPD enzymes and (ii) plantsare produced which are substantially tolerant to representative examplesof herbicide Formula 2 such as:

3-[[2-methyl-6-(trifluoromethyl)-3-pyridinyl]carbonyl]-bicyclo[3.2.1]-octane-2,4-dioneand/or

3-[[2-(ethoxymethyl)-6-(trifluoromethyl)-3-pyridinyl]carbonyl]-bicyclo[3.2.1]-octane-2,4-dioneand/or

3-[[2-(methoxyethoxymethyl)-6-(trifluoromethyl)-3-pyridinyl]carbonyl]-bicyclo[3.2.1]-octane-2,4-dione

R₁-R₆ are each individually selected from the group consisting of H,—C₁-C₄ alkyl, C₃-C₆ cycloalkyl, halogen, OH, SH, CN, —NH₂, —NHCOR,—CONHR, —COR, —SR, SOR, —SO₂R, NHR—SO₂R, —CO₂R, —NO₂, CF₃, —SF₅, OR, andCO₂H where R=C₁—C₆ alkyl or aryl optionally substituted with one or moresubstituents selected from the group consisting of halo or C₁-C₄ alkoxy.

Herbicidal HPPD inhibitors of Formula 3 include their agronomicallyacceptable salts. In preferred embodiments R₁ is SO₂Me, R₃ is CF₃ or Cland R₂, R₄ and R₅ are each H; in the case that R₃ is CF₃, the compoundis the active diketonitrile derivative of the herbicide isoxaflutole.According to particular preferred embodiments (i) polynucleotides of theinvention are selected to encode HPPD-inhibitor resistant HPPD enzymesand (ii) plants are produced which are substantially tolerant torepresentative examples of herbicide Formula 3 (or compounds which giverise to them) such as

5-cyclopropyl-4-(2-methylsulphonyl-4-trifluoromethylbenzoyl)-isoxazoleand/or

1-[2-(methanesulfonyl)-4-(trifluoromethyl)phenyl]-3-cyclopropyl-2-cyano-propane-1,3-dione

the former of these compounds is the herbicide isoxaflutole, the secondis its active derivative.

where Ar groups K and J are independently chosen from optionallysubstituted phenyl or optionally substituted heteroaryl. R₁-R₄ are eachindividually selected from the group consisting of —C₁-C₄ alkyl, C₃-C₆cycloalkyl, halogen, OH, SH, CN, —NH₂, —NHCOR, —CONHR, —COR, —SR, SOR,—SO₂R, NHR—SO₂R, —CO₂R, —NO₂, CF₃, —SF₅, OR, and CO₂H where R=C₁-C₆alkyl or aryl optionally substituted with one or more substituentsselected from the group consisting of halo or C₁-C₄ alkoxy. Optionalsubstituents for groups K and J include —C₁-C₄ alkyl, C₃-C₆ cycloalkyl,halogen, OH, SH, CN, —NH₂, —NHCOR, —CONHR, —COR, —SR, SOR, —SO₂R,NHR—SO₂R, —CO₂R, —NO₂, CF₃, —SF₅, OR, and CO₂H where R=C₁-C₆ alkyl oraryl optionally substituted with one or more substituents selected fromthe group consisting of halo or C₁-C₄ alkoxy.

Herbicidal HPPD inhibitors of Formula 4 include their agronomicallyacceptable salts. In some preferred embodiments Ar is substituted phenyland R₁₋₄ are each methyl. The said phenyl may have H at all positionsother than 2 and 4, which are then preferably substituted at position 2with NO₂, Me, OMe or Cl and at position 4 with SO₂Me, CN, OR or Cl whereR=C₁-C₆ alkyl or aryl optionally substituted with one or moresubstituents selected from the group consisting of halo or C₁-C₄ alkoxy.In a further preferred embodiment Ar is a substituted 2-pyridyl and R₁and R₂ are both H. The said pyridyl may have H at all positions otherthan 3 and 5, which are then preferably substituted at position 3 withR′ and at position 6 with CF₂H, CF₂CI or CF₃ and where R′ is Me,isopropyl, n-propyl, CH₂OMe, CH₂OEt, CH₂CH₂OMe or CF₃.

The present invention also provides an HPPD inhibitor resistant HPPDenzyme obtainable from Avena, Lolium, Chenchrus, Festuca, Eleusine,Brachiara or Sorghum plants.

The present invention further provides an HPPD inhibitor resistant HPPDenzyme having a sequence selected from the group consisting of SEQ IDNos. 8, 10, 12, 14, 16, 18 or 20 or a sequence that has, based on theClustal method of alignment and when compared along any given 150 aminoacid stretch of the alignment, at least 93% identity with the sequenceof SEQ ID Nos. 8, 10, 12, 14, 16, or 18 or the enzyme of SEQ ID No. 4 ora sequence that has, based on the Clustal method of alignment and whencompared along any given 150 amino acid stretch of the alignment, atleast 91% identity with the sequence of SEQ ID No. 4.

The skilled man is well aware of what is meant by the Clustal method ofalignment and reference to it is made in WO 00/32757.

The present invention also provides herbicide resistant plants whichcontain a heterologous polynucleotide which comprises a region whichencodes a triketone resistant HPPD, HPPD enzyme of the currentinvention.

The present invention further provides a method of selecting apolynucleotide which encodes a triketone inhibitor specific resistantHPPD inhibitor enzyme comprising screening a population of HPPD enzymeencoding sequences and selecting as those which encode an HPPD inhibitorresistant HPPD enzyme those sequences which encode an enzyme which incomparison with a control HPPD enzyme is either at least 2.5 orpreferably four fold more resistant to herbicides selected from FormulaI as compared to herbicides selected from Formula 3 or is at least 2.5or preferably four fold more resistant to herbicides selected fromFormula 2 as compared to Formula 4, wherein the said control enzyme isselected so as to exhibit substantially the same selection ofpolynucleotides as is obtained when the control enzyme is derived fromArabidopsis.

The present invention yet further provides a method of selecting apolynucleotide which encodes a syncarpic acid specific HPPD inhibitorresistant HPPD enzyme comprising screening a population of HPPD enzymeencoding sequences and selecting as those which encode resistant HPPDenzyme those sequences which encode an enzyme which in comparison with acontrol HPPD enzyme is at lease 2.5 or preferably four fold moreresistant to HPPD inhibitors selected from Formula 1 and 4, as comparedto Formula 1 and wherein the said control enzyme is selected so as toexhibit substantially the same selection of polynucleotides as isobtained when the control enzyme is derived from Arabidopsis. Thecontrol HPPD may be derived from a dicot—particularly Arabidopsis ortobacco, and the resistance of HPPD enzymes to herbicides may. bedetermined by measuring the rate of dissociation of the enzyme/herbicidecomplex.

The HPPD enzyme encoded by the selected polynucleotide may have a kcat/Km hydroxyphenylpyruvate value in the range from 0.10 to 5 s⁻¹ μM⁻¹ atpH 7.0, 25° C.

The present invention further provides a method for selectingpolynucleotides which comprise a region encoding an HPPDinhibitor-resistant HPPD enzyme which comprises screeningpolynucleotides comprising a region which encodes an HPPD enzyme andselecting as polynucleotides comprising a region encoding an HPPDinhibitor-resistant HPPD enzyme those which encode an enzyme capable offorming a complex with triketone herbicidal HPPD inhibitors selectedfrom Formula I and/or from Formula 2 wherein the dissociation of thesaid complex is governed by a dissociation constant (Kd), in water at pH7.0 and at 25 C, within the range from 1.0 to 30 nM, and wherein thedissociation of the said complex has a dissociation rate constant (Vff),in water at pH 7.0 and at 25 C, within the range from 4×10⁻⁵ to 2×1⁻³s⁻¹ and wherein said selected herbicidal HPPD inhibitors have at least aquarter of the herbicidal activity of mesotrione against dicot plants.

The present invention further provides a method for providing a plantwhich is tolerant to HPPD-inhibiting herbicides which comprisestransformation of plant material with a polynucleotide which comprises aregion which encodes an inhibitor resistant HPPD enzyme of the currentinvention as described above, or selectable according to any the methodsof the current invention described above, and regeneration of thatmaterial into a morphologically normal fertile plant, with the provisothat the HPPD sequence is not derived from Shewanella colwelliana, orPseudomonas fluorescens.

The polynucleotide may further comprise a region which encodes a proteincapable of targeting the HPPD encoded by the sequence to subcellularorganelles such as the chloroplast or mitochondria and the saidtargeting protein may have the sequence of (i) a chloroplast transitpeptide or (ii) a chloroplast transit peptide-N-terminal portion of achloroplast protein—chloroplast transit peptide.

The said polynucleotide may further comprise a sequence which encodes anHPPD-inhibiting herbicide degrading or otherwise detoxifying enzyme,and/or a protein otherwise capable of specifically binding to the saidHPPD-inhibiting herbicide.

The polynucleotide may further comprise a region which encodes (i) thetarget for a non-HPPD inhibitor herbicide and/or (ii) a non-HPPDinhibitor herbicide degrading or otherwise detoxifying enzyme and/or aregion encoding a protein capable of conferring on plant materialtransformed with the region resistance to insects, fungi and/ornematodes.

The said target or enzyme may be selected from the group consisting of acytochrome p450, a glutathione S transferase, glyphosate oxidase (GOX),phosphinothricin acetyl transferase (PAT),5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), acetolactatesynthase (ALS), protoporphyrinogen oxidase (PPGO) and phytoenedesaturase (PD) or mutagenised or otherwise modified forms thereof.

The present invention yet further provides a morphologically normalfertile whole plant obtained by any of the methods of the currentinvention which are described above.

The present invention further provides use of the polynucleotideselectable according to any of the methods of the current inventiondescribed above in the production of plant tissues and/ormorphologically normal fertile whole plants which are transgenic for theinhibitor resistant HPPD enzyme.

The present invention further provides a method of selectivelycontrolling weeds at a locus comprising crop plants and weeds, whereinthe plants are obtained by any of the methods of the current inventiondescribed above, wherein the method comprises application to the locusof a weed controlling amount of an HPPD inhibitor. The HPPD inhibitormay be selected from the group consisting of herbicides having theFormulae I to 4 as indicated above. A pesticide selected from the groupconsisting of an insecticide, a fungicide and a non-HPPD inhibitorherbicide may also be applied to the locus.

The present invention further provides use of the polynucleotideselectable according to any embodiment of the current inventiondescribed above in the production of a herbicidal target for the highthroughput in vitro screening of potential herbicides and in particularembodiments of this screening aspect of the invention the proteinencoding regions of the polynucleotide may be heterologously expressedin E. coli or yeast.

In one aspect, the current invention relates to methods for theselection of polynucleotides comprising a region which encodes HPPDenzymes exhibiting a level of inherent tolerance to certain herbicideswhich is useful for application in herbicide tolerant plants. As well asexhibiting a high level of inherent tolerance to a selected HPPDinhibitor (k_(off), Ki or Kd value) an HPPD enzyme encoded by apolynucleotide of the current invention may also, preferably, bepossessed of high stability and high catalytic activity where catalyticactivity is expressed by the parameter kcat/Km.

Methods for measuring the Km with respect to hydroxyphenylpyruvate ofHPPD enzymes are well known. However, hitherto, the relative instabilityof HPPD has precluded measurement of true, relatively undiminished kcatvalues. Thus in a further aspect, the invention relates to methods forthe selection of polynucleotides comprising a region which encode HPPDenzymes exhibiting kcat/Km values within a useful and determined range.

It will be appreciated that many methods well known to the skilled manare available for obtaining suitable candidate polynucleotides forscreening and selection which comprise a region encoding an HPPD from avariety of different potential source organisms including microbes,plants, fungi, algae, mixed cultures etc. as well as environmentalsources of DNA such as soil. These methods include inter alia thepreparation of cDNA or genomic DNA libraries, the use of suitablydegenerate oligonucleotide primers, the use of probes based upon knownsequences or complementation assays (for example, for growth upontyrosine) as well as the use of mutagenesis and shuffling in order toprovide recombined or shuffled HPPD-encoding sequences.

In certain embodiments of selection, polynucleotides comprisingcandidate and control HPPD encoding sequences are expressed in yeast, ina bacterial host strain, in an alga or in a higher plant such as tobaccoor Arabidopsis and the relative levels of inherent tolerance of the HPPDencoding sequences screened according to a visible indicator phenotypeof the transformed strain or plant in the presence of differentconcentrations of the selected HPPD inhibitors. Dose responses andrelative shifts in dose responses associated with these indicatorphenotypes (formation of brown colour, growth inhibition, herbicidaleffect etc) are conveniently expressed in terms, for example, of GR50(concentration for 50% reduction of growth) or MIC (minimum inhibitoryconcentration) values where increases in values correspond to increasesin inherent tolerance of the expressed HPPD.

It will be appreciated that many combinations of host organism,indicator phenotype and control HPPD would achieve a similar scope ofselection and these are contemplated within the scope of the currentinvention. For example, in a relatively rapid assay system based upontransformation of a bacterium such as Ecoli, each HPPD encoding sequencemay be expressed, for example, as a DNA sequence under expressioncontrol of a controllable promoter such as the lacZ promoter and takingsuitable account, for example by the use of synthetic DNA, of suchissues as codon usage in order to obtain as comparable a level ofexpression as possible of different HPPD sequences. Such strainsexpressing polynucleotides comprising alternative candidate HPPDsequences may be plated out on different concentrations of the selectedherbicides in, optionally, a tyrosine supplemented medium and therelative levels of inherent tolerance of the expressed HPPD enzymesestimated on the basis of the extent and MIC for inhibition of theformation of the brown, ochronotic pigment.

In variations of the method the cells may be permeabilized or,particularly in the case of yeast, be strains having disabled pumps inorder to minimise the effects of differential uptake and export of HPPDinhibitors into and out of the cell. In a preferred variation of themethod bacterial cells are grown almost to stationary phase in a liquidmedium, exposed to selected herbicides for a short period of one hour orless, resuspended in a similar volume of fresh medium and the rate ofdevelopment of pigment monitored. In a further preferred methodcandidate HPPD expressing sequences are transferred to a shuttle vectorand, similar to above, are each expressed at a comparable level, butthis time in a suitable Pseudomonas species such as Pseudomonasfluorescens 87-89 capable of being transformed and of growing ontyrosine as sole carbon source. Preferably the endogenous HPPD gene ofthe host Pseudomonas line is knocked out, for example, byrecombinational insertion of an antibiotic marker gene. Pseudomonaslines each transformed to express an alternative resistant HPPD enzymeare grown on different concentrations of selected HPPD inhibitors andthe inherent resistance of the expressed HPPD sequence in respect ofeach HPPD inhibitor estimated upon the basis of the concentrationnecessary to prevent growth on a medium containing tyrosine as solecarbon source.

One skilled in the art will recognise that there exist many potentialvariants of these methods for selecting polynucleotides which wouldachieve essentially the same selection result and which are contemplatedwithin the scope of the current invention. In general, suchmicroorganism-based methods of selection are suitable for achieving arelatively high throughput of candidate polynucleotides and areparticularly suited to initial pre-screening. However, because ofpotential problems with the acuity of discrimination arising from thedifferential uptake and metabolism of selected herbicides and,furthermore, because the very high inherent potencies of many herbicidalHPPD inhibitors may limit the theoretical resolution of methods basedupon MIC values, it is preferable to also use further embodiments of theselection method of the current invention.

In further particularly preferred aspects of the methods of the presentinvention for screening and selecting polynucleotides comprising asequence encoding preferentially inhibitor resistant HPPD enzymes,candidate polynucleotides are transformed into plant material,regenerated into morphologically normal fertile plants which plants arethen measured for differential tolerance to selected HPPD-inhibitorherbicides. Many suitable methods for transformation using suitableselection markers such as kanamycin, binary vectors such as fromAgrobacterium and plant regeneration as, for example, from tobacco leafdiscs are well known in the art. Optionally, a control population ofplants is likewise transformed with a polynucleotide expressing thecontrol HPPD. Alternatively, an untransformed dicot plant such asArabidopsis or Tobacco can be used as a control since this, in any case,expresses its own endogenous HPPD. The average, and distribution, ofherbicide tolerance levels of a range of primary plant transformationevents or their progeny to herbicidal HPPD inhibitors selected fromFormula 1, Formula 2, Formula 3 and/or Formula 4 are evaluated in thenormal manner based upon plant damage, meristematic bleaching symptomsetc. at a range of different concentrations of 15 herbicides. These datacan be expressed in terms of, for example, GR50 values derived fromdose/response curves having “dose” plotted on the x-axis and “percentagekill”, “herbicidal effect”, “numbers of emerging green plants” etc.plotted on the y-axis where increased GR50 values correspond toincreased levels of inherent tolerance of the expressed HPPD. Herbicidescan suitably be applied pre-emergence or post-emergence.

Polynucleotides of the invention are selected as those where, determinedon the basis of the their effects on plants, the ratio of the inherenttolerance of the expressed HPPD to an inhibitor selected from Formula Ior 2 to that for an HPPD inhibitor selected from Formula 3 or Formula 4(Ri2/R34) is either, at least about 2.5 and preferably four fold greaterthan, or, at least about 2.5 and preferably four fold less than the sameratio determined in respect of the same pair of selected compounds forthe control HPPD. It will be appreciated that many combinations ofhigher or lower plant, indicator phenotype, transformation method,assessment method and control HPPD would achieve a similar scope ofselection and are contemplated within the scope of the currentinvention. Transient expression of the test HPPD genes in suitablegreen, transiently transformable green tissues such as mesophyll cellprotoplasts or tobacco leaves is also optionally used in order toprovide a more rapid means of selection. Suitable methods for suchtransient transformation of tissues are well known in the art andinclude, for example, leaf infiltration, vacuum infiltration andinfection with Agrobacterium or bombardment of target tissues withDNA-coated particles.

In these transient assay methods, treated tissue is, for example,suitably transferred to media containing a range of concentrations ofselected herbicides after about 0. 1-7 days after transformation andassessed for visible signs of bleaching after a further 1-5 d. In orderto provide an internal control to allow for differences in transientexpression, constructs used for transformation may also comprise a genesuch as GUS which expresses a readily quantifiable product. Whilst apreferred method, a limitation of methods based upon stabletransformation of plants for polynucleotide selection include therelatively large number of events (preferably greater than 25) required,time-scale of several months required to turn around data and thefurther breeding, segregation analysis and testing of furthergenerations which is ideally required to resolve biologicalvariabilities and to make comparisons between the alternative HPPD genesexpressed from different constructs.

In further particularly preferred embodiments of the selection methodsof the present invention polynucleotides comprising a candidate regionencoding an HPPD inhibitor resistant HPPD enzyme are selected on thebasis of in vitro measurements of the comparative inherent resistancelevels of the expressed candidate and control HPPD enzymes.

The particular combination of in vitro methods and criteria used hereinare new. It is found here that active principles of HPPD herbicideswhich either are, or which have the potential to be, commercially usefultend also to be such potent inhibitors of HPPD enzymes that Ki valuesand other kinetic parameters useful for comparing the inherentresistance of HPPD enzymes cannot be derived from steady state enzymekinetic or IC50 based enzyme assay methods as have generally beendescribed in the HPPD literature.

Apparent IC50 values may generally be determined by arbitraryexperimental parameters such as the concentration of enzyme used in theassay and the time allowed for reaction. Neither, even given the use ofmore appropriate methods, has it hitherto been known that processeshitherto described to partially or completely purify HPPD cause suchdamage to the enzyme as to alter the values of kinetic parameters and tosuch an extent as to confound useful comparison between the inherenttolerances of HPPD enzymes. In particular, the effect of a highproportion of the enzyme molecules being damaged and of diminishedcatalytic activity (expressed on a per active site basis) as a result ofpart purification is to reduce the measured apparent strength of HPPDbinding interactions with inhibitors.

By way of a non-limiting illustration of the in vitro methods preferredherein, the HPPD sequences may conveniently be expressed in a yeast orin E.coli using, for example, expression from a T7 polymerase promoteror other such suitable methods which are well known in the art. Suitableextracts for in vitro experiments may, for example, be prepared by cellbreakage, removal of cell debris and insoluble proteins bycentrifugation and exchange of the fraction containing the expressedsoluble HPPD enzyme into a suitable buffer. The, thus prepared extractmay, optionally, be beaded frozen and stored at liquid Nitrogentemperature until required for use. Control HPPD enzymes are likewiseprepared. Preferably, the handling and partial purification of the HPPDis minimised since, as mentioned above, it is found here, that mostmethods of attempting to purify or, optionally reconstitute with ironions, result in losses of activity and inhibitor binding capacity whichmay obfuscate the desired comparisons between inherent resistance andactivity level.

Optionally, the enzyme may be part-purified in the presence ofinhibitors such as structure VIII (see later) which have a stabilisingeffect but which do not bind so tightly that they are difficult tosubsequently remove. In vitro measurements are suitably carried outusing, for example, E.coli extracts wherein the HPPD expressed from thetransgene constitutes, for example, 0.25-10% of the total solubleprotein. In a particular embodiment of the methods for selection ofpolynucleotides, the inherent resistance of expressed HPPD enzymes isevaluated in vitro on the basis of the rate of dissociation of theenzyme/herbicide complex (k_(off) value) and/or, according to thedissociation constant (Kd ) of the enzyme/herbicide complex.

Thus, in one aspect of the invention there is provided a method forselecting polynucleotides which comprise a region encoding anHPPD-inhibitor resistant HPPD enzyme which comprises screening apopulation of HPPD encoding sequences and selecting as those whichencode an HPPD-inhibitor resistant HPPD enzyme those which encode anenzyme able to form a complex with herbicidal HPPD inhibitors selectedfrom Formula I and/or Formula 2 wherein, in water at pH 7.0 and at 25 C,the dissociation of the said complex is governed by a dissociationconstant (Kd) in the range 1-30 nM and/or a dissociation rate constant(k_(off)) in the range from 4×10⁻⁵ to 2×10⁻³ s⁻¹ and wherein theselected HPPD-inhibitor has at least a quarter of the herbicidalactivity of mesotrione versus dicot plants. Activity versus dicot plantsrefers here to herbicidal activity averaged over a range of 6 or more ofthose dicot weed and crop species usually used in screens used forcompound discovery in the agrochemical community. Herbicidal activityversus dicot plants also refers here to that activity which is due tothe inhibitor per se rather than due to some, potentially moreherbicidal, metabolite of it which may be formed in planta or otherwise.

In a further aspect of the invention there is provided a method forselecting polynucleotides which comprise a region encoding anHPPD-inhibitor resistant HPPD enzyme which comprises screening apopulation of HPPD encoding sequences and selecting as those whichencode an HPPD-inhibitor resistant HPPD enzyme those which encode anenzyme able to form a complex with herbicidal HPPD inhibitors selectedfrom Formula 3 and/or Formula 4 wherein, in water at pH 7.0 and at 25 C,the dissociation of the said complex is governed by a dissociationconstant (Kd) in the range 1-30 nM and/or a dissociation rate constant(k_(off)) in the range from 4×10⁻⁵ to 2×10⁻³ and wherein the selectedHPPD-inhibitor has at least a quarter of the herbicidal activity ofmesotrione versus dicot plants.

In a yet further aspect there is provided a method of selecting apolynucleotide which encodes an HPPD-inhibitor resistant HPPD enzymecomprising screening a population of HPPD enzyme encoding sequences andselecting as those which encode an HPPD-inhibitor resistant HPPD enzymethose sequences which encode an enzyme which, in comparison with acontrol enzyme, exhibits at least a 2.5 fold and preferably greater thana 4 fold difference in inherent resistance to HPPD inhibitors selectedfrom Formula I and/or 2 as compared to Formula 3 and/or 4 and whereinthe said control enzyme is selected so as to exhibit substantially thesame selection of polynucleotides as is obtained when the control enzymeis the wild type HPPD derived from Arabidopsis. To illustrate furtherwhat is meant by this and also what is meant by some of the terms usedin the in vitro-based methods of selection of the current invention whatfollows relates to a non limiting example wherein the selectedpolynucleotide expresses HPPD from Avena sativa and wherein the controlHPPD sequence is from Arabidopsis. The definitions and basis ofselection used in this illustration apply analogously to the selectionof other polynucleotides which encode other HPPD enzymes and which areselectable according to either the same or other in vitro methods of theinvention. According to this example, a polynucleotide comprising asequence encoding, in this case, the HPPD enzyme from Avena sativa, isselected as resistant when, in comparison with a control HPPD enzyme, inthis case from Arabidopsis, the Avena HPPD enzyme is found to be morethan 2.5 fold resistant to herbicidal inhibitors selected from Formula Iand/or Formula 2 as compared to herbicides selected from Formula 3and/or Formula 4. By this is meant, that, assayed under identicalconditions (e.g at 25 C in 50 mM Bis-Tris-propane buffer at pH 6.5 or7.0 containing either <4% or 25% v/v glycerol and either<2 or 20-25 mMsodium ascorbate) and preferably assayed using the same method, side byside on the same day:

(a) HPPD inhibitors selected from Formula 3 or 4 dissociate more slowlyfrom the complex formed with HPPD derived from Avena than doHPPD-inhibitors selected from Formula 1 or 2, to the extent that theratio of the value of koff (as illustrated in the scheme below) for thecompound selected from Formula 1 or Formula 2 to the value of k_(off)for the compound selected from Formula 3 or 4 (k_(off)12/k_(off)34) isat least 2.5 fold and, preferably more than 4 fold greater than thelikewise derived ratio observed in respect of dissociation of the samepair of selected inhibitors from the, likewise obtained, Arabidopsiscontrol enzyme.

Herbicidal inhibitors of HPPD are found here to have generally lowvalues of k_(off) (in the range less than 0.0003 s⁻¹, often less than0.000001 s⁻¹). It will be appreciated that many suitable methods knownin the art for determining such low k_(off) values are suitable forworking the current invention. These include measuring rates of exchangeof radio or otherwise labelled inhibitors either with or away from theenzyme inhibitor complex. For example enzyme inhibitor complexes canreadily be prepared by incubating HPPD preparations with labelled orunlabelled inhibitor and then, after suitable periods, optionallyrapidly separating the thus formed enzyme inhibitor complex from excessinhibitor by any suitable method such as ultrafiltration, binding tofilters or exchange down a gel filtration column. Exchange reactionswith the, thus prepared enzyme inhibitor complex is then initiated byaddition of, as appropriate, excess labelled or unlabelled inhibitor.HPPD preparations suitable for use in the methods of the currentinvention are relatively unpurified, buffer-exchanged, supernatantfractions of spun crude lysates of E.coli strains engineered to expressthe HPPD enzyme of interest at a level of, typically, about 0.25-10% ofthe total soluble protein. Many methods such as radiometric,fluorimetric, NMR, fluorescence depolarisation, EPR, Mossbauer, UV/VISspectrophotometry etc. or phonon resonance can, in principle, be used tomonitor the enzyme/ligand exchange reactions and, particularly in thiscase where the enzyme contains an iron atom at the ligand binding site.Optionally, the monitoring method may be continuous (as, for example,with scintillation proximity/bead-based methods) or, discontinuous,based upon collection of data at various timepoints wherein samples areremoved and the bound and unbound label components rapidly separated andquantitated.

Values of koff can suitably be calculated by computer fitting based uponnumerical integration of the exchange data along with information on theactive-site concentration of HPPD and upon Vn values obtained asdescribed below. In crude extracts of, for example, Arabidopsis HPPD itis routinely found that approximately 20-30% of bound mesotrioneexchanges relatively rapidly (t ½<˜30-40 min for dissociation ofmesotrione at 25 C, pH 7.0 in 20-25% v/v glycerol) whereas 70-80%,presumed here to correspond to the bulk of genuine fully active enzymeexchanges slowly (t ½˜4 d for dissociation of mesotrione at 25 C, pH 7.0in 20-25% v/v glycerol). This presumption is supported by (1) theobservation that further enzyme handling associated with activity lossleads to a relative increase in the proportion of the rapid exchangingfraction and (2) the observation that the fraction does not, on theother hand, vary according to the time of the complex formation (10 s to24 h) and, is not, therefore, a kinetically trapped intermediate in thebinding reaction. In any event, k_(off) values are always herecalculated from the rate of the major slow exchange reaction. It will beappreciated that within the scope of the current invention many methodsof making the desired kinetic comparisons are possible without explicitor rigorous determination of off rates but, based upon the sameunderlying principle, will achieve the same selection result.

Or: b) herbicidal inhibitors selected from Formula 3 or 4 bind, relativeto the substrate HPP, more tightly to HPPD derived from Avena than doherbicides selected from Formula I or 2, to the extent that the ratio ofthe value of Kd (Kd=k_(off)/k₀, illustrated in the scheme below) for thecompound selected from Formula I or Formula 2 to the value of Kd for thecompound selected from Formula 3 or 4 (Kdl2/Kd34) is at least 2.5 foldand, preferably more than 4 fold greater than the likewise derived ratioobserved in respect of binding of the same pair of selected inhibitorsfrom the, likewise obtained, Arabidopsis control enzyme.

The method for determining k_(off) values is outlined supra. In someembodiments of the method, Kd is determined by also determining thevalue of k_(on), the rate constant governing the rate of formation ofthe complex of HPPD with inhibitor wherein Kd=k_(off)/k_(on). Suitableenzyme kinetic methods for deriving values of k_(on) are based upon therate of onset of enzyme inhibition over a range of concentrations ofinhibitor and of substrate. Suitable methods combine, for example, theHPLC assay for HPPD described by Viviani et al 1998 (Pestic. Biochem.Physiol., 62, 125-134) which assay can be started with addition ofenzyme and data points collected over the first minute or so ofreaction, standard methods for measurements of the value of the Km forhydroxyphenylpyruvate and methods of kinetic analysis/calculation asdescribed for example by Schloss J. V. (1989) in “Target sites ofHerbicide Action”(Boger P., and Sandmann G. eds) CRC Press Boca.

Alternatively estimates of k_(on) values can be determined more directlyby mixing HPPD with radio or otherwise labelled herbicide inhibitor andmonitoring the progress of the binding reaction, optionally by rapidlyisolating the enzyme inhibitor complex and/or by any one of a number ofmethods (for example fluorimetry, EPR, NMR, radiodetection etc). Forexample, the reaction with HPPD may be started by addition ofradiolabelled herbicide, allowed to proceed for a series of differenttimes and rapidly quenched by addition and mixing with a large excess ofunlabelled inhibitor. In this case the extent of binding at differenttimes may, for example, be monitored by using ultrafiltration, bindingto filters or gel filtration to separate radiolabel-bound to HPPD fromunbound label which fractions can then each be quantitated byscintillation counting.

When measuring k_(on) via such measurements of physical binding it isimportant to note that the binding of most compounds versus some HPPDenzymes appears biphasic with half the sites binding quickly and thenthe remaining binding then occurring relatively very slowly. In suchcases, it is the rapid initial binding phase, usually corresponding torate constants in the range 0.1-4×10⁵ M⁻¹ s⁻¹, which provides therelevant rate constant. This corresponds to the value obtained fromusing enzyme assay-based methods since although only half the sites areinitially bound, on the same time-scale essentially all of the HPPDcatalytic activity is inhibited. It will be appreciated that within thescope of the current invention many more or less rigorous methods ofmaking the desired kinetic comparisons, are possible which may notinvolve explicit determination of off rates and on rates but, based uponthe same underlying principles, achieve the same selection result.

Thus, for example, in a preferred and relatively high throughput method,relative Kd values, which are all that is required for determining therequired ratios of the Kd values of the selected HPPD inhibitors, areestimated indirectly via competition with the binding of a knownstandard or other ‘surrogate’ ligand. Such a surrogate ligand could beany molecule including a peptide, optionally, initially selected from aphage display library, an RNA aptamer or an antibody fragment. In apreferred embodiment it is a labelled HPPD inhibitor. Therefore,structure I or IV or V may be used as a labelled standard, andexperiments set up where the relative Kd values of the selected HPPDinhibitors are evaluated on the basis of their ability to compete withand decrease the amount of labelled standard bound to the test orcontrol HPPD.

STRUCTURE I. 2-(Nitro-4-methanesulphonylbenzoyl)-cyclohexane-1,3-dione

STRUCTURE II.3-[[2-methyl-6-(trifluoromethyl)-3-pyridinyl]carbonyl]-bicyclo[3.2.1]-octane-2,4-dione

STRUCTURE III.3-[[2-(ethoxymethyl)-6-(trifluoromethyl)-3-pyridinyl]carbonyl]-bicyclo[3.2.1]-octane-2,4-dione

STRUCTURE IV.1-[2-(methanesulfonyl)-4-(trifluoromethyl)phenyl]-3-cyclopropyl-2-cyano-propane-1,3-dione

STRUCTURE V.2-[2-nitro-4-chlorobenzoyl]-4,4,6,6-tetramethylcyclohexane-1,3,5-trione

STRUCTURE VI.2-[2-methyl-4-cyanobenzoyl]-4,4,6,6-tetramethylcyclohexane-1,3,5-trione

STRUCTURE VII.3-[[2-methyl-6-(trifluoromethyl)-3-pyridinyl]carbonyl]-4,4,6,6-tetramethylcyclohexane-1,3,5-trione

STRUCTURE VIII.2-[cyclopropylcarbonyl]-5-ethyl-4-methanesulfonyl-4-methyl-cyclohexane-1,3-dione

Ideally, in order to obtain the best approximations to equilibrium Kdvalues the competition binding reactions of HPPD plus standard and testinhibitors should be left to equilibrate at, for example, 25° C. for aslong as possible to reach equilibrium and, preferably, days beforesampling and evaluation via gel filtration or binding to anitrocellulose filter etc of the amount of label which is unbound andwhich is bound to HPPD. It will be understood that such reactions may beleft for shorter periods due to limitations in enzyme stability andthat, as reaction times are made shorter, the values obtained become andmore weighted to reflect differences in k_(on) values rather than pureKd values. It will be understood that, within the scope of the presentinvention, a great variety of alternative technologies such as thatbased upon Luminex fluoresence bead technology or Scintillationproximity counting could potentially be used, for example to avoid theneed for a step to separate bound from unbound label, and provideessentially the same result. Using such methods the determination ofrelative Kd values can also be converted to a microtitre plate formatand be useful not only for the selection of polynucleotides comprisingregions which encode HPPD enzymes but also for the discovery of smallmolecule inhibitors as potential leads for new chemical herbicides.

In a yet further aspect, the invention comprises a method for,optionally, further selecting polynucleotides which encodeinhibitor-resistant HPPD enzymes having a high catalytic activity bywhich is meant a kcat/Km hydroxyphenylpyruvate value in the range from0.10 to 5 s⁻¹ μM-1 at pH 7.0, 25° C. Assays and measurements of Km arecarried out using published methods such as the HPLC assay of Viviani etal 1998 (Pestic. Biochem. Physiol., 62, 125-134). Assay time coursescurve off rapidly and, using such stopped methods, it is important tomake sufficient initial rate measurements at suitably short times and tofit the data obtained appropriately to obtain rate estimates. SuitableHPPD preparations which retain most of the enzyme in a fully active formare, for example, rapidly prepared as relatively crude,buffer-exchanged, supernatant fractions of spun crude lysates of E. colistrains engineered to express the HPPD enzyme of interest at a level of,typically, about 0.2-10% of the total soluble protein.

In order to obtain kcat, the Vmax value (mol of HGA formed/s), obtainedfrom experiments in which substrate concentration is varied, is dividedby the concentration of enzyme active sites. There are many methods ofdetermining active-site concentration. Herbicides such as that ofstructure I, IV or V bind very tightly to the active site of HPPDenzymes and, optionally labelled, make suitable active site probesuseful for the determination of active-site concentration. Thus, forexample, from titrations of extract containing an unknown concentrationof active sites of HPPD versus a fixed concentration of labelledinhibitor, it is possible to describe a graph of extract dilution versusthe amount of bound label and to thereby derive the concentration ofinhibitor binding sites or ‘active sites’. Many methods are suitable formonitoring the binding reaction including for example, use ofradiolabels, NMR, EPR, Biacore (Pharmacia) etc.

Because the binding of some HPPD inhibitors is biphasic it is importantto carry out the binding titration carefully and to vary the inhibitorand time since, in some cases the result obtained will be closer to a‘half sites’ rather than a full quantitation of active siteconcentration. The binding reaction used for the titration needs, as faras possible, to be left to reach equilibrium as modified by practicalconsiderations of enzyme stability. It will be appreciated that withinthe scope of the present invention many, more or less rigorous methodsof making the desired kinetic comparisons are possible which may notinvolve explicit determination of kcat/Km but, based upon the sameunderlying principles, achieve the same result in terms of ranking therelative efficacies of polynucleotides comprising regions encoding anHPPD enzyme. For example, kcat and hence kcat/Km values may be derivedby using antibodies raised to SDS PAGE purified HPPD polypeptides inorder to quantitate the amount of HPPD polypeptide in active crudeextracts using quantitative fluorescent Western or ELISA type assays.However, methods based upon quantitation of polypeptide are blind towhether or not the material represents active enzyme and, for thisreason, the methods for the determination of kcat based upon inhibitorbinding are preferred because, for inhibitors resembling catalyticreaction intermediates, the retention of this tight-binding capabilityis synonymous with the retention of catalytic function. As HPPI) isfurther purified and loses more activity the damaged enzyme still bindslabelled inhibitor but, as the activity diminishes, an increasingproportion of this binding becomes weaker and more rapidly exchanging.Therefore in a preferred embodiment of the method, the fraction ofinhibitor binding sites which are in relatively rapid exchange arediscounted in the calculation of kcat. Thus, for example, in crudeextracts of Arabidopsis HPPD it is routinely found that, of the totalmeasured binding capacity for mesotrione (Structure I), approximately20-30% exchanges rapidly (t ½˜30-40 min for dissociation of mesotrioneat 25 C, pH 7.0 in 25% v/v glycerol) whereas 80%, presumed here tocorrespond to active enzyme exchanges slowly (t ½˜4 d for dissociationof mesotrione at 25 C, pH 7.0 in 20% v/v glycerol). Thus, in this case,Kcat may be based upon an active site determination calculated as˜80% ofthe total measured binding capacity, although the values cited in thisapplication do not take that potential adjustment into account.

In one aspect the present invention provides HPPD-inhibitor resistantHPPD enzymes which are not derived from maize, wheat or barley and whichare characterised by the ability of the enzyme to form a complex withmesotrione wherein the dissociation of the said complex in water at pH7.0 and at 25 C is governed by a dissociation constant (Kd) having avalue in the range from 1.0 to 30 nM and/or wherein the dissociation ofsaid complex is governed by a rate constant (k_(off)) having a value inthe range from 4×10⁻⁵ to 2×10⁻³. In a further aspect, the saidHPPD-inhibitor resistant enzyme is further characterised by having akcat/Km value in the range from 0.1 to 5 s⁻¹ μM⁻¹ and, more preferably,in the range from 0.8 to 5 s⁻¹ μM⁻¹.

In a further aspect an HPPD-inhibitor resistant HPPD enzyme has an aminoacid sequence selected from the group consisting of SEQ ID NOs. 8, 10,12, 14, 16, 18 or 20 or a sequence that has, based on the Clustal methodof alignment and when compared along any given 150 amino acid stretch ofthe alignment, at least 93% identity with the sequence of SEQ ID NOs. 8,10, 12, 14, 16, or 18 or an HPPD inhibitor resistant HPPD enzyme of SEQID NO:4 or a sequence that has, based on the Clustal method of alignmentand when compared along any given 150 amino acid stretch of thealignment, at least 91% identity with the sequence of SEQ ID NO:4.

The structures of HPPD inhibitors referred to in the specification andin some of the preferred embodiments of the invention are as follows.Note that wherever structures are drawn in a keto form that thesestructures can also exist in an enolic form and that all of these andall other tautomeric forms are also intended.

According to particular preferred embodiments (i) polynucleotides of theinvention are selected to encode HPPD-inhibitor resistant HPPD enzymesand ii) plants are produced which are substantially tolerant torepresentative examples of herbicide Formula 4 such as

2-[2-nitro-4-chlorobenzoyl]-4,4,6,6-tetramethylcyclohexane-1,3,5-trioneand/or

2-[2-methyl-4-cyanobenzoyl]-4,4,6,6-tetramethylcyclohexane-1,3,5-trioneand/or

3-[[2-methyl-6-(trifluoromethyl)-3-pyridinyl]carbonyl]-4,4,6,6-tetramethylcyclohexane-1,3,5-trione

The structures of the specific HPPD inhibitors referred to as numberedStructures I to VIII have already been described. According toparticular preferred embodiments (i) polynucleotides of the inventionare selected to encode HPPD-resistant HPPD enzymes and (ii) plants areproduced which are substantially tolerant to one or more of thesestructures. Note that wherever structures are drawn in a keto form thatthese structures can also exist in an enolic form and that all of theseand all other tautomeric forms are also intended.

It will be appreciated that the transformed plants, and the thustransformed plant material, of the present invention are tolerant orresistant to multiple herbicides within the groups of HPPD inhibitorsrepresented by Formula 1, 2, 3 and 4 as well as to HPPD-inhibitingherbicides outside of these groupings such as5-methyl-2-(2-Chloro-3-ethoxy-4-methanesulphonylbenzoyl)-cyclohexane-1,3-dione.

It will also be appreciated that those embodiments which are tolerant toHPPD inhibitors selected from Formula I and 2 will generally be lesstolerant or resistant to herbicides, representative of Formula 3 and 4such as structure V. Conversely, those embodiments which are tolerant toHPPD inhibitors selected from Formula 3 and 4 will be generally lesstolerant or resistant to herbicides, representative of Formula 1 and 2such as structure I (mesotrione). Where the embodiments are transgenicplants, herbicide may be applied either pre- or post emergence inaccordance with the usual techniques for herbicide application.

The invention still further provides protein encoded by the presentlydisclosed polynucleotides and a vector comprising these polynucleotidescomprising a HPPD sequence under expression control of a promoterderived from the gene encoding the small subunit of rubisco, a cestrumviral promoter, an actin promoter, a polyubiquitin promoter, the FMV35Spromoter, a plastocyanin promoter, a histone promoter, the CaMV35Spromoter and the GST1 promoter. In a further preferred embodiment, wherethe said plant is a monocot, the HPPD sequence is under expressioncontrol of a maize polyubiquitin promoter or a cestrum viral promoter.In a yet further preferred embodiment, where the said plant is a dicotcrop plant, the HPPD sequence is under expression control of anarabidopsis small subunit of rubisco promoter, an arabidopsis actinpromoter or a cestrum viral promoter.

The transformed plant material of the invention may be subjected to afirst HPPD inhibitor -such as a triketone herbicide and visuallyselected on the basis of a colour difference between the transformed andnon transformed material when subjected to the said herbicide. Thenon-transformed material may become and stay white when subjected to theselection procedure, whereas the transformed material may become whitebut later turn green, or may remain green, likewise, when subjected tothe said selection procedure. Plant transformation, selection andregeneration techniques, which may require routine modification inrespect of a particular plant species, are well known to the skilledman. In preferred embodiments of the selection method the said DNA(which distinguishes transformed from non-transformed plants) comprisesa region selected from the group consisting of SEQ ID NOs 3, 7, 9, 11,13, 15, 17 and 19 or it comprises a region which encodes an HPPD, whichregion is complementary to one which when incubated at a temperature ofbetween 60 and 65° C. in 0.3 strength citrate buffered saline containing0.1% SDS followed by rinsing at the same temperature with 0.3 strengthcitrate buffered saline containing 0.1% SDS still hybridises with asequence selected from the group consisting of SEQ ID NOs. 3, 7, 9,11,13,15,17 and 19.

When the test and inventive sequences are double stranded the nucleicacid constituting the test sequence preferably has a T_(M) within 10° C.of that of the sequence selected from the group consisting of SEQ ID NOs3, 7, 9, 11, 13, 15, 17 and 19. In the case that the test and thesequence selected from the group consisting of SEQ ID NOs.3, 7, 9, 11,13, 15, 17 and 19 are mixed together and are denatured simultaneously,the T_(M) values of the sequences are preferably within 5° C. of eachother. More preferably the hybridisation is performed under relativelystringent conditions, with either the test or inventive sequencespreferably being supported. Thus either a denatured test or inventivesequence is preferably first bound to a support and hybridisation iseffected for a specified period of time at a temperature of between 60and 65° C. in 0.3 strength citrate buffered saline containing 0.1% SDSfollowed by rinsing of the support at the same temperature but with 0.1strength citrate buffered saline. Where the hybridisation involves afragment of the sequence selected from the group consisting of SEQ IDNOs. 3, 7, 9, 11, 13, 15, 17 and 19 the hybridisation conditions may beless stringent, as will be obvious to the skilled man.

In the case that the polynucleotide encodes more than one protein, eachprotein encoding region may be under the transcriptional control of aplant operable promoter and terminator. It may be desired to target thetranslation products of the polynucleotide to specific sub-cellularcompartments within the plant cell, in which case the polynucleotidecomprises sequences encoding chloroplast transit peptides, cell walltargeting sequences etc. immediately 5′ of the regions encoding the saidmature translation products.

Translational expression of the protein encoding sequences containedwithin the said DNA sequence may be relatively enhanced by includingknown non translatable translational enhancing sequences 5′ of the saidprotein encoding sequences. The skilled man is very familiar with suchenhancing sequences, which include the TMV-derived sequences known asomega, and omega prime, as well as other sequences derivable, interalia, from the regions 5′ of other viral coat protein encodingsequences, such as that of the Tobacco Etch virus. Further preferred 5′untranslated regions include those derived from, for example, the genesencoding rubisco or glucanase.

The polynucleotides of the invention may be modified in that encodedmRNA instability motifs and/or fortuitous splice regions are removed,or, for example, dicot preferred codons are used so that expression ofthe thus modified sequence in a dicot plant yields substantially similarprotein having a substantially similar activity/function to thatobtained by expression of the unmodified sequence in the organism inwhich the protein encoding regions of the unmodified sequence areendogenous. In a further embodiment of the modified sequence the degreeof identity between the modified sequence and a sequence endogenouslycontained within the said dicot plant and encoding substantially thesame protein is less than about 70%.

The present invention also provides a-morphologically normal fertilewhole plant which is transgenic for a DNA sequence, which is not derivedfrom maize, wheat or barley and which is selectable according to themethods of the current invention such that it comprises a region whichencodes an HPPD-inhibitor resistant HPPD enzyme, preferably of highstability and having a kcat/Km value in the range from 0.10 to 5.0 s⁻¹mM⁻¹, more preferably in the range from 0.8 to 5.0 s⁻¹ mM⁻¹ which, incomparison with a control HPPD enzyme derived from Arabidopsis, is atleast 2.5 fold and, preferably, greater than 4 fold more resistant toherbicides selected from Formula 1 or Formula 2 than to herbicidesselected from Formula 3 or Formula 4. Alternatively, the plant istransgenic for a similarly derived sequence which is selected on thebasis that it comprises a region which encodes an HPPD-inhibitorresistant HPPD enzyme able to form a complex with herbicidal HPPDinhibitors selected from Formula 1 and/or Formula 2 wherein, in water atpH 7.0 and at 25 C, the dissociation of the said complex is governed bya dissociation constant (Kd) in the range 1-30 nM and/or a dissociationrate constant (k_(off)) in the range from 4×10⁻⁵ to 2×10⁻³ s⁻¹ andwherein the selected HPPD-inhibitor has at least a quarter of theherbicidal activity of mesotrione versus dicot plants. In furtherembodiments the said plant is transgenic in respect of a polynucleotidecomprising a DNA sequence which encodes an HPPD-inhibitor resistant HPPDenzyme derived from a plant or, more particularly, derived from amonocot plant or, yet more particularly, from a rice, Brachiaria,Chenchrus, Lolium, Festuca, Setaria, Eleusine, Sorghum or Avena species.In yet further embodiments the said DNA comprises a sequence selectedfrom the group consisting of SEQ ID NOs 3, 7, 9, 11, 13, 15, 17 and 19.

Plants transformed according to the present inventive method include butare not limited to, field crops, fruits and vegetables such as canola,sunflower, tobacco, sugar beet, cotton, maize, wheat, barley, rice,sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon,mangelworzel, potato, carrot, lettuce, cabbage, onion, etc. Particularlypreferred genetically modified plants are soya spp, sugar cane, pea,field beans, poplar, grape, citrus, alfalfa, rye, oats, turf and foragegrasses, flax and oilseed rape, and nut producing plants insofar as theyare not already specifically mentioned . In a particularly preferredembodiment of the method the said plant is a dicot, preferably selectedfrom the group consisting of canola, sunflower, tobacco, sugar beet,soybean, cotton, sorghum, tomato, mango, peach, apple, pear, strawberry,banana, melon, potato, carrot, lettuce, cabbage, onion, and isparticularly preferably soybean. In further preferred embodiments thesaid plant is maize or rice. Preferably the plant of the invention issoybean, rice or maize. The invention also includes the progeny of theplant of the preceding sentence, and the seeds or other propagatingmaterial of such plants and progeny.

The present invention also includes the use of the DNA sequencereferenced above in the production of plant tissues and/ormorphologically normal fertile whole plants wherein i) the tolerance ofplants to herbicidal HPPD inhibitors is increased, wherein the increaseis greater to HPPD inhibitors selected from Formulae 1 or 2 is greaterthan that to HPPD inhibitors selected from Formulae 3 or 4, or whereinthe increase is greater to HPPD inhibitors selected from Formulae 3 or 4is greater than that to HPPD inhibitors selected from Formulae 1 or 2and/or (ii) which contain relatively elevated levels of lipid solubleanti-oxidants when compared with non-transgenic such tissues or plants.“Lipid soluble antioxidants” include suitable plastoquinones,α-tocopherols and carotenoids such as the precursors of vitamin A, forexample.

The present invention still further provides a polynucletide comprisingtranscriptional enhancers and an HPPD inhibitor resistant HPPD enzymeunder expression control of its autologous promoter which enzyme isidentifiable according to presently disclosed method. Preferably thesaid HPPD enzyme has the sequence depicted in SEQ ID NO:4. Also includedin the invention are plant cells which have been transformed with apolynucleotide sequence which encodes an HPPD inhibitor resistant HPPDenzyme, characterised in that the HPPD encoding sequence is selectableaccording to presently disclosed methods and/or is derived from anorganism selected from the group consisting of Shewenella Colwellina,Vibrio vulnificus, Steptomyces avermitilis and Coccidiodes immitus.Preferably, when the cells are dicot cells the promoter region used tocontrol expression of the HPPD encoding sequence is derived from thesmall sub-unit of rubisco, and when the cells are monocot cells thepromoter region is derived from the maize poly-ubiquitin gene.

The invention will be further apparent from the following descriptiontaken in conjunction with the associated sequence listings.

EXAMPLE 1 Cloning of Full and Partial Length 4-HPPD Sequences from Avenaand Other Monocot Species

Total RNA is prepared from five-day-old Avena Sativa, Brachiariaplatyphylla, Cenchrus echinatus, Lolium ridgidum, Festuca arundinacea,Setaria faberi, Eleusine indica and Sorghum sp. seedlings using themethod of Tri-Zol extraction (Life Technologies). RT-PCR is performed oneach of the RNA samples using the One-step RTPCR kit (Invitrogen) inconjunction with primers HPPD5 (SEQ ID NO:32) and HPPD4R (SEQ ID NO:31). The products obtained are cloned into vector pCR2. ITOPO(Invitrogen) and the cloned products sequenced using standard M13forward and reverse primers. The sequences obtained are given (orcomprised within), for example, SEQ ID NOs. 3, 7, 9, 11, 13, 15, 17 and19. Messenger RNA is obtained, for example, from Avena sativa using theOligotex mRNA purification system (Qiagen). The 5′ end of, for example,the A. sativa HPPD gene is identified using 5′ RACE, performed using theGene Racer kit (Invitrogen) with gene specific primers (GSP) HPPD RT2(SEQ ID NO:21) and HPPD RT4 (SEQ ID NO:22). The 3′ end of the gene isidentified by 3′ RACE, performed using Themoscript RT (LifeTechnologies) with oligo dT primer DT30 (SEQ ID No 23), followed by PCRwith GSP HPPD3 (SEQ ID NO:24) and primer DTR (SEQ ID NO:25). Allmethodologies are performed according to protocols provided by thevarious stated manufacturers. Products obtained from the 5′ and 3′ RACEreactions are cloned into pCR 2.1 TOPO (invitrogen) and the clonedproducts sequenced using universal M13 forward and reverse primers withan automated ABI377 DNA sequencer. Primers 5′ Avesa1 (Seq ID NO:26) and3′ Avesa (Seq ID NO:27) are designed to the translation initiation andtermination codons of the HPPD gene (SEQ ID NO:3) respectively. Bothprimers are used in conjunction with the One-step RTPCR kit (Qiagen) toobtain full length coding sequences. Products obtained are cloned intopCR 2.1 TOPO, sequenced, and identified as 4-HPPD by comparison withsequences known in the art.

EXAMPLE 2 Heteroloious Expression of the Pseudomonas fluorescens,Arabidopsis and wheat 4-HPPD genes in E.coli

The sequences of the Pseudomonas fluorescens strain 87-79 (see WO98/20144), Arabidopsis (see WO 97/2728) and Wheat 4-HPPD (see WO00/32757) genes are all known in the art. All three genes are obtainedby RT-PCR using primers incorporating suitable restriction enzyme sitesin order to allow their cloning into suitable E. coli over-expressionvectors, such as the pET (Novagen) series and, for example, as describedin Example 3. Heterologous expression of the Pseudomonas HPPD gene in E.coli is also described in WO 98/20144, the contents of which areincorporated herein by reference and heterologous expression ofArabidopsis HPPD in E.coli is also described in Garcia et al in PlantPhysiol (1999) 119, 1507-1516) the contents of which are incorporatedherein by reference.

EXAMPLE 3 Heterologous Expression of the Avena sativa 4-HPPD Gene in E.coli

The full length A. sativa HPPD gene is excised from the pCR 2.1 TOPOvector, described in example 1, using Nde 1 and Bam H1, and ligated intosimilarly restricted pET-24a (Novagen). This vector is then transformedinto E. coli BL2 1 (DE3) codon+RP cells (Stratagene). Suitable hoststrains such as BL21(DE3) or other DE3 lysogens harbouring the saidvector express quantities of HPPD enzyme sufficient to provide for theiruse in high through put screening to identify alternative 4-HPPDinhibitors. Authenticity of the transformed line is confirmed by PCR,plasmid recovery, and restriction analysis. HPPD purified from the saidtransformed host strain (for example by SDS gel electrophoresis andexcision of the band) may be used in the provision of antisera for theanalysis of plants transformed with a polynucleotide encoding 4-HPPD.

EXAMPLE 4 Heterologous Expression of Pseudomonas 4-HPPD in Tobacco

The Pseudomonas fluorescens gene from strain 87-79 (SEQ ID NO: 1) isedited by PCR to include 5′ Nco1 and 3′ Kpn1 sites. This product is thenligated into pMJB1. pMJB1 is a pUC19 derived plasmid which contains theplant operable double enhanced CaMV35S promoter; a TMV omega enhancerand the NOS transcription terminator. A schematic representation of theresulting plasmid is shown in FIG. 2 of WO 98/20144. The expressioncassette, comprising the double enhanced 35S promoter, TMV omega leader,4-HPPD gene and nos terminator, is excised using Hind III/Eco R1(partial Eco R1 digest) and cloned into similarly digested pBIN 19 andtransformed into E. coli TOP 10 competent cells.

DNA is recovered from the E. coli and used to transform Agrobacteriumtumefaciens LBA4404, and transformed bacteria selected on media containrifampicin and kanamycin. Tobacco tissue is subjected toAgrobacterium-mediated transformation using methods well described inthe art and, optionally, as is described elsewhere herein. Transformedshoots are regenerated from kanamycin resistant callus. Shoots arerooted on MS agar containing kanamycin. Surviving rooted explants arere-rooted to provide approximately 50 kanamycin resistant transformedplants.

EXAMPLE 5 Heterologous Expression of Wheat HPPD Sequence in Tobacco

The wheat HPPD gene is obtained by RT-PCR using primers TAHPPDNde (SEQID NO:28) contains an Ndel site at translation initiation codon orTAHPPDSph (SEQ ID NO:29) contains Sph 1 site at the translationinitiation codon and TAHPPDBam (SEQ ID No.30) contains a BamH1 site attranslation stop codon. The PCR products are cloned into pCR 2.1, andsequenced to confirm authenticity. The Nde1:BamH 1 product is ligatedinto the vector pMCJA, which is a derivative of pMJB1 (WO 98/20144) andcontains an Ndel site at the translation initiation codon rather thanNco1. The Sph1:BamH1 products are ligated into vector ATSSU1, a pUCderived vector comprising the Arabidopsis small sub-unit of rubiscopromoter and nos terminator or ATSSU2, a pUC derived vector comprisingthe Arabidopsis small sub-unit of rubisco promoter, an optimised transitpeptide and the nos terminator. These gene expression cassettes are allthen transferred into suitable binary vectors such as BIN19 (and relatedvectors) and termed TAHPPD1 (FIG. 1), TAHPPD2 (FIG. 2) and TAHPPD3 (FIG.3) respectively. These constructs were all transformed intoAgrobacterium tumefaciens strain LBA4404, which in turn was used totransform tobacco, using methodology described previously.

Explants (i.e. a leaf plus short segment of stem containing theauxiliary bud) are placed into MS agar (+3% sucrose) containing variousconcentrations of mesotrione (see above) from 0.02 to 2 ppm. In tobacco,for example, untransformed explants are fully bleached at 0.02 ppm. Theydo not recover following prolonged exposure to the herbicide. In theseparticular experiments, only the shoot that develops from the bud isbleached, the leaf on the explanted tissue remains green.

A number of the PCR+ve transformed plants tolerate 0.1 ppm of mesotrione(about 5 times the level which causes symptoms on wild-type tobacco, forexample) with no indication of bleaching. They root normally and arephenotypically indistinguishable from untransformed plants. A sub-set ofthe transformants is tolerant to concentrations of>0.2 ppm yieldingplants looking normal and rooting well in the presence of herbicide.Some of the transformed plants can be initially bleached when subjectedto the herbicide at the said higher concentrations, but on prolongedexposure they progressively “green up” and “recover”.

A subset of the said herbicide resistant transgenic plants are treatedwith the known herbicide Isoxaflutole [5-cyclopropyl-4-(2-methylsulphonyl-4-trifluoromethylbenzoyl)-isoxazole]or, alternatively, the syncarpic acid of structure VI. The said plantsare, relative to untransformed controls, resistant to all the herbicidesbut are, however, substantially less resistant to isoxaflutole, theactive principle of which is the diketonitrile (structure IV) aherbicide of Formula 3 or to structure VI a herbicide of Formula 4 thanthey are to mesotrione, a herbicide of Formula I thus clearly indicatingthat the plants are not fully cross resistant to multiple classes of4-HPPD inhibitor, which although having the same mode of action are ofdistinct structural types.

EXAMPLE 6 Heterologous Expression of the Avena sativa 4-HPPD Gene inTobacco

The Avena sativa 4-HPPD gene contained within the pCR 2.1 TOPO vector(example 1) is excised from the vector using Nde1 and BamH1 and ligatedinto similarly digested pMCJA. The structure of the resulting vector isshown schematically in FIG. 4.

The 4-HPPD plant expression cassette is then ligated in to the binaryvector pBinl9 restricted with Hind III and EcoR1 and transformed into E.coli TOP10 cells (Invitrogen). This binary vector is then transformedinto tobacco using methods well known in the art and, for example, asdescribed elsewhere herein.

A subset of the said herbicide resistant transgenic plants are treatedwith the known herbicide Isoxaflutole[5-cyclopropyl-4-(2-methylsulphonyl-4-trifluoromethylbenzoyl)-isoxazole]or, alternatively, the syncarpic acid of structure VI. The said plantsare, relative to untransformed controls, resistant to all the herbicidesbut are, however, substantially less resistant to isoxaflutole, theactive principle of which is the diketonitrile (structure IV) aherbicide of Formula 3 or to structure VI a herbicide of Formula 4 thanthey are to mesotrione, a herbicide of Formula I thus clearly indicatingthat the plants are not fully cross resistant to multiple classes of4-HPPD inhibitor, which although having the same mode of action are ofdistinct structural types.

EXAMPLE 7 Production of DNA for Plant Transformation

Linear DNA, suitable for use in bombardment plants transformation, isproduced by digesting a vector containing the plant expression cassettewith a suitable restriction enzyme(s) to excise the said cassette, whichis then purified on an agarose gel and isolated using a Biotrap(Schleicher and Schuell). For agrobacterium transformation of soybean orcorn the plant expression cassette is subcloned into binary vectors asdescribed in examples 12 and 13.

EXAMPLE 8 In Planta Screening and Selection of PolynucleotidesComprising a Region Encoding an HPPD-Inhibitor Resistant HPPD

Plants are 1) untransformed tobacco plants variety Samsun expressing theendogenous tobacco (control) HPPD gene, 2) tobacco plants transformed toexpress Pseudomonas HPPD according to the examples herein and 3) tobaccoplants transformed to express Wheat HPPD also according to the examplesherein.

Large numbers of plants are grown from seed in small pots in theglasshouse to the 5-7 leaf seedling stage and sprayed with a range ofdoses, suitably from 0.0 to 2000 g/ha, of different HPPD-inhibitorherbicides selected from compounds of Formula 1, 2, 3 and 4. Treatmentsare suitably Formulated in, for example, deionised water+0.5%Turbocharge™ surfactant or, alternatively, 50% acetone/water and appliedat 2001/ha to a dozen or more replicates of each line and at each sprayrate (where the plants were T1 plants (selfed progeny of primarytransformants)and still segregating at normally 1:2:1) or, where,homozygous, 3-6 plants. The extent of visible damage in terms ofbleaching of meristems and leaves, eventual necrosis and stunting ofgrowth relative to unsprayed controls is assessed at˜1 and 3 weeks aftertreatment. Data from susceptible segregants are excluded from theanalysis. In summary, results obtained are as follows.

Control (untransformed) plants are˜1-2 fold more susceptable ofisoxaflutole and˜2-4 fold more susceptible of Structure VI, StructureVII or Structure V than of mesotrione, Structure I. Structure II is ofsimilar potency to isoxaflutole. Plants expressing wheat HPPD are 10-40fold less susceptible of mesotrione than they are Structure VI orStructure V and also 4-10 fold less susceptible of mesotrione than theyare of isoxaflutole. Plants expressing wheat HPPD are 4-15 fold lesssusceptible of Structure II than they are of Structure V, Structure VIor Structure VII. Plants expressing pseudomonas fluorescens HPPD are 2-6fold more tolerant of isoxaflutole, Structure V, Structure VI orStructure VII than they are of mesotrione or Structure II.

The results demonstrate, inter alia, plants, comprising a testpolynucleotide comprising a region encoding a wheat HPPD, which are, forexample, >16× more tolerant of structure I, mesotrione, a compoundselected from Formula 1 than they are, for example, of structure VI, acompound selected from Formula 4 whereas, for untransformed controlplants, the respective difference in tolerance in respect of the samecompounds is<4. The ratio of the two, Formula 1/Formula 2 toleranceratios in respect of test and control plants is, therefore, atleast>16/4 which is>2.5 and also>4. Therefore, according to the method,the polynucleotide comprising a region encoding wheat HPPD is screenedvia transgenesis, regeneration, breeding and spray testing of tobaccoand, according to these results, selected as one which encodes an HPPDinhibitor resistant HPPD enzyme. The results also demonstrate, plants,comprising a test polynucleotide comprising a region encoding aPseudomonas HPPD, which are, for example, >2-4 × more tolerant ofStructure V, Structure VI or Structure VII, compounds selected fromFormula 4 than they are, for example, of Structure I, mesotrione, acompound selected from Formula 1 whereas, for untransformed controlplants, the respective difference in tolerance in respect of the samecompounds is<˜0.3-0.5. The ratio of the two, Formula 4/Formula 1tolerance ratios in respect of test and control plants is, therefore, atleast>2/0.5 which is>2.5 and also>4. Therefore, according to the method,the polynucleotide comprising a region encoding a Pseudomonas HPPD isscreened via transgenesis, regeneration, breeding and spraying oftobacco and thereby selected as one which encodes an HPPD inhibitorresistant HPPD enzyme.

EXAMPLE 9 In Vitro Screening and Selection of Polynucleotides ComprisingRegions which Encode HPPD Enzymes having kcat/Km Values in the Rangefrom 0.10-5/uM/s

Crude extracts of recombinant E.coli strains expressing, different testHPPD sequences from, for example, Arabidopis Wheat, Avena Sativa,Pseudomonas etc and as described elsewhere in the examples are prepared.The recombinant clones are introduced into BL21 (DE3) a codon-optimisedRP strain of E.coli supplied by Stratagene. The HPPD protein isexpressed in this strain following addition of 1 mM IPTG to thefermenter medium (e.g. LB medium supplemented with 100 μg/ml Kanamycin).The recombinant protein of the correct predicted mass is identified (i)on the basis of Coomassie staining of SDS gels of cell extracts and sideby side comparison with Coomassie-stained gels of extracts of similarE.coli cells transformed with an empty pET24a vector and (ii) by westernanalysis using a polyclonal antibody previously raised to HPPDpolypeptide cut out and eluted from an SDS PAGE gel. Typically, 25 g wetweight of cells are washed in 50 ml of 0.1 M Hepes/KOH buffer at pH 7.5.Following low-speed centrifugation, the cell pellet is resuspended in 50ml of the same buffer. Cells are evenly suspended using a glasshomogenizer and then disrupted at 10000 psi using a Constant Systems(Budbrooke Rd, Warwick, U.K.) Basic Z cell disrupter. The crude extractis centrifuged at˜40,000 g for˜2 h and the pellet discarded. Clearsupernatant fraction is then exchanged into the same buffer downSephadex G25 and the, thus prepared extract either used fresh, or beadedinto liquid Nitrogen and stored at −80C until use. Typically, judgedfrom Coomassie dye stained SDS PAGE gels, extracts contained 1-4% of thesoluble protein as HPPD. Typically protein concentrations are in therange 15-30 mg/ml and specific activities, based upon using the HPLCassay at 25 C and a substrate concentration of 100 μM HPP are in therange 50-300 nmol of HGA produced/min/mg of protein.

The titre of enzyme inhibitor binding sites in each enzyme preparationis quantitated as follows. A set of reactions are set up in eppendorfcentrifuge tubes at ice temperature. A range of volumes of extract,typically from 0 to 50 μl are diluted to a final volume of 250 pl inreaction buffer and the reaction in each tube initiated by addition of afixed amount of radiolabelled inhibitor. Reaction buffer is suitably, 50mM Bis-Tris-propane buffer at pH 7.0 containing, (freshly made) 25 mMsodium ascorbate and 2-3.8 mg/l of high-purity catalase (Sigma C315-50,000 units/mg of protein). Optionally, reaction buffer also contains25% v/v glycerol. Radiolabelled HPPD inhibitor is suitably labelled with14C at between 0.5 and 3 GBq/mmol and the inhibitor is suitable selectedfrom, mesotrione, the diketonitrile derived from isoxaflutole or5-methyl-2-(2-Chloro-3-ethoxy-4-methanesulphonylbenzoyl)cyclohexane-1,3-dione. The fixed concentration of radiolabel in eachtube is suitably set at 0.1-0.4 μM HPPD inhibitor. In the case of someinhibitor/HPPD enzyme pairs, reactions can be run for i) a relativelyshort period at 25 C, suitably for 5-15 minutes or ii) for a longperiod, overnight at 4 C followed by 3-5 h at 25 C in order to achieve,respectively, i) half of the sites or ii) substantially completeoccupancy of sites. Those skilled in the art will appreciate that thisis a matter of experiment. At the end of the period samples, typically0.2 ml, of the reaction are taken and rapidly chromatographed down a˜2ml Pharmacia NAP5′ gel filtration column and separated intoprotein-containing (0.8 ml) and protein-free (3 ml) fractions. The twofractions are divided jnto scintillation vials, scintillation fluidadded and the number of counts in each fraction totalled up. Thosecounts in the protein-containing fraction represent radiolabelledinhibitor bound to protein and those counts in the protein-free fractionrepresents unbound inhibitor. The purity and radiochemical specificactivity of radiolabelled inhibitor is known. Therefore, on theassumption that the inhibitors bind to HPPD in proportions ofapproximately either I or 0.5 per catalytic sites/protein monomer theconcentration of inhibitor binding sites and therefore of catalyticsites in the extract can be calculated. Such stoichiometry would beexpected for inhibitors which are active-site directed and which mimiccatalytic intermediates. By way of further example, the results of sometypical experiments are given below. 0, 2, 5, 10, 20, 40 and 60 μlaliquots of a 20 mg protein/ml Arabidopis HPPD extract were eachincubated with 10 jl (196 Bq) of ¹⁴C mesotrione (final concentration ˜17μM at˜1. 12 GBq/mmol) in a final volume of 240 μl of 50 mM Bis-Trispropane buffer at pH 7.0 and at 25 C containing 25 mM sodium ascorbateand 2-3.8 mg/l of high-purity catalase (Sigma C3155-50,000 units/mg ofprotein) for 24 h at ice temperature and then at 25 C for -3.5h. Pilotexperiments indicated that, under such conditions, mesotrione bindingwould be near complete, the initial -50% of binding occurring rapidly,the remaining 50% more slowly. 200 oil samples of each reaction weretaken and chromatographed down a NAP5 column in order to separateprotein-bound from unbound radiolabel and the two fractions counted. Theresults obtained are depicted in TABLE 1 and expressed in FIG. 5 but,after suitable corrections, with bound dps converted to the μMconcentration of bound label in the original reaction.

In agreement with TLC and NMR studies, Table 1 indicates that 95% of theradiolabel is mesotrione (as defined by its binding to HPPD) with˜4-5%corresponding to radiolabel contaminants which are not mesotrione andwhich do not exhibit tight binding to HPPD. The concentration ofmesotrione binding sites in the undiluted Arabidopsis extract is -12.2jiM. The polypeptide Mr of Arabidopsis HPPD is -50 kD; thus it can becalculated that HPPD consititutes 3% of the protein of the originalcrude Arabidopsis HPPD extract. TABLE 1 Titration of arabidopis HPPDversus ¹⁴C mesotrione Volume of extract (μl) bound 14C (dps) total 14C(dps) % bound 0 0.3 169.9 0.0 2 22.95 168.9 13.6 5 56.48 178.23 31.7 10113.3 173.1 65.5 20 152.3 166.25 91.6 40 157.6 168 93.8 60 158.7 168.194.4

It will be apparent to those skilled in the art that essentially thesame methods can be used to measure the titre of inhibitor-binding sitesin extracts of other HPPD enzymes. Thus, for example, an extract of HPPDat fro m wheat at 24 mg protein/ml is determined to contain˜20+/−4 μMbinding sites for ¹⁴C-labelled diketonitrile or¹⁴C5-methyl-2-(2-Chloro-3-ethoxy-4-methanesulphonylbenzoyl)-cyclohexane-1,3-dione. For wheat, the first inhibitor molecule binds more quickly,the second more slowly and the error range is higher than forArabidopsis HPPD because the wheat enzyme is somewhat less stable andloses its binding capacity more quickly. Wheat HPPD (and indeed all HPPDenzymes) is stabilised by inhibitors and most stabilised by thoseinhibitors which bind the tightest to it. Such inhibitors are the bestchoice for measuring the titre of inhibitor binding sites

The HPLC assay for HPPD activity and the determination of kcat and Km isconducted as follows. Assay buffer is 105 lM (or as appropriate)hydroxyphenylpyruvate (HPP) is freshly made up in 50 mM Bis-Tris-propanebuffer at pH 7.0. Dilution buffer is 50 mM Bis-Tris-propane buffer at pH7.0 containing 25 mM sodium ascorbate and 3.8 mg/l of bovine catalase(Sigma C3155 -50,000 units/mg). HPPD enzyme, freshly unfrozen fromstorage is kept at ice temperature and diluted, also at ice temperature,to an appropriate concentration in dilution buffer (typically, 2-8 μM)before use. Assays are started by addition of 5 μl of diluted enzyme to100 μl of assay buffer at 25 C in an eppendorf centrifuge and stopped,at a series of times between 0 and 90s by addition of 20 il of 25%perchloric acid and whirlimixing. 80-100 μl of the contents of eacheppendorf tube is transferred to an HPLC vial prior to separation byReverse Phase HPLC. For HPLC, 40 pl is loaded at 1.5 ml/min onto an AquaC18 5μ75×4.6 mm column (silica is endcapped) equilibrated with 5.5%acetonitrile, 0.1% trifluoroacetic acid (buffer A) using an HP 1100 HPLCsystem. The column is eluted, also at 1.5 ml/min, using a 2 minute washwith buffer A, followed by a 2 min wash with a 30/70 mixture of bufferA/100% acetonitrile followed by a further 3.5 minute wash with buffer A(in between uses the column is stored in 65% acetonitrile/water). Theelution of HGA (homogentisic acid) and HPP(hydroxyphenylpyruvate) ismonitored using a UV flow cell and quantitated via integration of peakabsorbance at 292 nm. HGA elutes at around 2 minutes and HPP eluteslater. A standard set of concentrations of HGA are used to provide astandard curve in order to calibrate the UV absorbance of the HGA peakversus HGA concentration.

The assay is used to provide estimates of the Km and Vmax values oftypical HPPD preparations. For Km determinations it is important toobtain near initial rate data which, for stopped assays, becomes morecritical at lower substrate concentrations. Thus it is important to takea number of time-points for each substrate concentration and to useearly time point data at low substrate concentrations. An example of anexperiment to determine the Km and Vmax value for the wheat HPPD isprovided in TABLE 2. TABLE 2 Wheat HPPD assayed at different times (s)and HPP concentrations Data are amounts of HGA formed (pmol). The stockwheat HPPD extract (18 μM) was diluted 30 fold. The assay, final volume105 μl contained 5 μl of diluted HPPD. 5 15 25 35 TIME (s) HPP concn. μM3 44 129 182 207 6 — 186 282 389 8 55 220 347 462 12 68 245 417 556 20113 352 479 777 60 133 427 700 963

The Km value for the wheat HPPD with respect to the substrate HPP isabout 10.1+/−1.5 μM . Vmax is 33.5+/−4 pmol/s. From the active-sitetitration the concentration of wheat HPPD in the assay is calculated tobe 31.5 nM corresponding to 3.1 pmol. of enzyme sites in 105 Ill. Thekcat and kcat/Km values for wheat HPPD can therefore be calculatedas˜11/s and˜1.1/s/μM, respectively. Similarly, it is determined that thekcat, Km and kcat/Km values of Arabidopsis HPPD are˜4.65/s, 3.5 μM and1.3/s/μM, respectively and also that the kcat, Km and kcat/Km values ofw/t Pseudomonas fluorescens HPPD are—5.04/s, 32 μM and 0.16 l s/μM,respectively. The mutant form of Pseudomonas fluorescens HPPD having atryptophan at position 336 is found to have a kcat/Km value at least 3fold reduced relative to that of the wild-type (kcat/Km<˜0.05/s/μM). Itcan be further calculated that the specific activities of the purewild-type Pseudomonas (subunit Mr˜40223), Arabidopsis (subunit Mr˜46486)and wheat enzymes (subunit Mr 48923) at 25 C and with saturatingsubstrate are, at least, 7.13, 5.7 and˜13.5 μmol/min/mg protein whichare values much higher than previously known for HPPD enzymes; thesevalues increase yet further by 20-30% when it is further taken intoaccount that 20-30% of the binding to the ‘fast exchanging’ fraction ofsites (vide infra) which quite possibly represents binding to damagedenzyme, or, sites otherwise unrelated to catalytic activity.

Therefore, according to this example a polynucleotide comprising aregion encoding, for example, a wheat HPPD is screened via a methodcomprising heterologous expression in a bacterium, preparation of anextract containing the expressed HPPD in an active form, determinationof the active site concentration through titration versus atight-binding active-site directed inhibitor and performing enzymeassays at a range of substrate concentrations. It is selected as apolynucleotide, useful in the context of the current invention, whichencodes a suitably resistant HPPD enzyme because the value of kcat/Kmcalculable from the data so obtained is˜1.0/s/μM at pH 7.0, 25° C. whichis within both the claimed range of 0. I to 5 s⁻² μM⁻² and the preferredrange of 0.8 to 5 s⁻¹ μM⁻¹.

EXAMPLE 10 In Vitro Screening and Selection of PolynucleotidesComprising Regions which Encode HPPD-Inhibitor Resistant HPPD EnzymesBased Upon Measurement of the Relative and Absolute Values of RateConstants Governing the Dissociation of Enzyme/Inhibitor Complexes

Crude extracts of recombinant E.coli strains expressing, in the onecase, a control HPPD (from Arabidopsis) and, in the other, one or more atest HPPD sequences are prepared as described in the preceding example.The titre of active sites and enzyme activity are also defined andmeasured as described in the preceding examples.

The dissociation rates (off rates) of inhibitors selected from Formula 1and/or 2 and/or 3 and/or 4 from the complexes that they form with testand control HPPD enzymes are suitably measured in a number of ways.Thus, for example, the rates of dissociation of mesotrione , a compoundselected from Formula 1 and of the diketonitrile of isoxaflutole, acompound selected from Formula 3 from their respective complexes with,test, wheat HPPD and with, control, Arabidopsis HPPD are compared. Themethod and results from a typical test are as follows.

¹⁴C mesotrione is of specific activity 1.12 GBq/mmol. This is˜95% pureradiochemically by TLC and based upon the proportion of materialtight-binding to HPPD. Arabidopsis and, (test), wheat HPPD are eachdiluted to a concentration of˜0.45 and 0.54 μM, respectively into 1.6 mlof 50 mM Bis-Tris propane buffer at pH 7.0 containing 25% glycerol, 25mM sodium ascorbate and 3 mg/l of bovine catalase (Sigma C3155˜50,000units/mg) containing 0.46 mM ¹⁴C mesotrione and left to react at 25 Cfor 2.5-3 h. Following this initial binding reaction, exchange reactionsare initiated by addition of cold mesotrione to a final concentration of60 μM and 200 μl aliquots removed at various times to rapidchromatography down a NAP5 gel filtration column equilibrated in 50 mMBTP at pH 7 containing 0.1 M KCl, separation into fractions containingprotein -bound or free radiolabel and liquid scintillation counting.Results are summarised in FIG. I in which in control experiments, whereno cold mesotrione is added, the inhibitor remains substantially bound.

The concentration of bound mesotrione (vertical axis) at zero time(˜0.35 μM) in FIG. 1 is somewhat less than either the concentration ofArabidopsis enzyme (˜>0.43 μM) or, in the case of wheat which was inexcess, of the inhibitor (˜0.46 μM). This is because, after 2.5 to 3 hat 25C binding is not fully complete (˜0.5 equivalents of mesotrionebind quickly, the remainder binds slowly) and the rate is somewhatslowed by the presence of 25% v/v glycerol. It is also apparent that˜25%of the mesotrione bound to Arabidopsis HPPD exchanges relatively rapidlywhereas the rest exchanges slowly. In crude extracts of Arabidopsis aswell as other HPPD enzymes it is routinely found that approximately20-30% of bound mesotrione exchanges relatively rapidly (t ½˜30-40 minfor dissociation of mesotrione from Arabidopsis HPPD at 25C, pH 7.0 in20-25% v/v glycerol) whereas 70-80%, presumed here to correspond to thebulk of genuine fully active enzyme exchanges slowly (t ½˜4 d fordissociation of mesotrione at 25C, pH 7.0 in 20-25% v/v glycerol). Thisis supported by 1) the observation that further enzyme handlingassociated with activity loss leads to a relative increase in theproportion of the rapid exchanging fraction and 2) the preliminaryobservation that the relative proportion of the fast exchanging fractiondoes not, on the other hand, vary according to the time of the bindingreaction (10 s to 24 h) and, is not, therefore, a kinetically trappedintermediate in the binding reaction. The notion that the fastexchanging fraction merely represents damaged enzyme is furthersupported by the observation that the proportion of the rapidlyexchanging fraction is diminished or not observed when experiments arecarried out with an excess of HPPD. The origin of the fast exchangingfraction is not entirely clear and remains open to speculation.Nevertheless, here, for practical purposes, koff values are always herecalculated from the rate of the major slow exchange reaction. Suchk_(off) values are calculated by computer modelling to obtain a best fitof the data to the computed progress of an EI+I* ← → EI*+I exchangereaction governed by four rate constants (but where the two k_(on)values are assumed to be the same as eachother as are the two k_(off)values) and using k_(on) values as independently determined in thefurther examples (the accuracy of k_(on) values, not, in any case,generally being critical for such fits). It is clear that mesotrionedissociates rapidly from the test, wheat, HPPD but much more slowly fromthe, control, Arabidopsis HPPD. The data are best fit to koff values formesotrione of 3.8×10⁻⁴/S from wheat HPPD and 2.0×10⁻⁶/s from ArabidopsisHPPD (k_(on) values in the two cases, in the presence of glycerol, being7×10⁴/s/M and 1.1×10⁵/s/M, respectively).

Not all test inhibitors are readily available in a radiolabelled form. Amore general method of measuring off rates is to first form the complexwith unlabelled test inhibitor, to rapidly exchange the complex free ofexcess unlabelled inhibitor and to then start the exchange reaction byaddition of an excess of a standard labelled inhibitor, the kineticproperties of which are already known. The reaction is then monitoredand the relative proportions of bound and unbound label determined atvarious times. It is found that k_(off) values so obtained frommonitoring the forward rate of exchange of labelled inhibitor bindingonto HPPD are, as expected, generally very close to the values obtainedfrom monitoring the reverse, which is the exchange of labelledinhibitors from the complex with HPPD. The latter is the method ofchoice when the rate of exchange is fast (t ½<3 h).

Apart from ¹⁴C mesotrione (structure I) (specific activity 1.12 GBq/mmolas used here), ¹⁴C structure III (specific activity 1.036 GBq/mmol asused here) and ¹⁴C5-methyl-2-(2-Chloro-3-ethoxy-4-methanesulphonylbenzoyl)-cyclohexane-1,3-dione (1.81 GBq/mmol as used here) are suitably used as standardinhibitors. Preferably whichever standard inhibitor is found to bind thetightest (has the lowest Kd value) to a given HPPD is used as thestandard inhibitor in respect of that HPPD for the evaluation of the Kdand k_(off) values of unlabelled inhibitors. The method and resultswhich follow illustrate the method for measuring the k_(off) values ofunlabelled inhibitors. Arabidopsis HPPD is diluted to 2.76 μM in 50 mMBis-Tris propane buffer at pH 7.0 containing 25 mM sodium ascorbate, 2mg/l of bovine catalase (Sigma C3155˜50,000 units/mg) and 20 μM of testinhibitor, in this case, mesotrione or structure II. This initialbinding reaction with unlabelled inhibitor is left overnight at icetemperature and then for 2-3 h at 25 C. 200 μl is then quickly exchangedfree of unbound inhibitor by gel filtration down a Pharmacia NAP5 columnand dilution into 1.5 ml of 50 mM Bis-Tris propane buffer at pH 7.0containing 25 mM sodium ascorbate and 2 mg/l of bovine catalase (SigmaC3155˜50,000 units/ mg). The exchange reaction is started by addition of80 μl of ¹⁴C mesotrione to a final concentration of 1.75 μM and aliquotsremoved at various times for rapid gel filtration down a NAP5 columnequilibrated in 50 mM BTP at pH 7 containing 0.1 M KCl, separation intoprotein-bound and protein-free fractions and liquid scintillationcounting. Results are summarised in FIG. 2.in which triangles representdata from unlabelled mesotrione, circles represent data from structureII. In control experiments, where the enzyme is pre-incubated witheither no unlabelled inhibitor or, preferably, with a weak inhibitorsuch as in structure VIII, the radiolabelled inhibitor is fully boundwithin a period of 5-6 h (to a concentration of 0.385 μM in theparticular example of the experiment depicted in FIG. 2) and remainssubstantially fully bound for a period of days (the amount bounddeclining by˜5-7%/d corresponding to gradual deterioration of theArabidopsis HPPD enzyme). Concentrations of bound on the vertical axisare normalised with respect to the control values in order to take intoaccount this gradual loss of binding capacity. The graphs obtained arethe inverse of the type depicted in FIG. 1. Again, the data areconsistent with—20% of the HPPD binding sites being in relatively rapidexchange with an initial phase of more rapid binding occurring first. Inaddition, in the controls, binding equilibrium is not full establisheduntil after 4-6 h. k_(off) values are always here calculated from therate of the major slow exchange reaction, which occurs after thisperiod. Such k_(off) values are calculated by computer modelling toobtain a best fit of the data to the computed progress of an EI+J ← →EJ+I exchange reaction governed by four independent rate constants andusing k_(on) values as independently determined in the further examples.Thus, for example, from the data in FIG. 2 it is clear that structure IIdissociates more rapidly from Arabidopsis HPPD than does mesotrione. Thedata are best fit to k_(off) values, obtained in this case without 25%v/v glycerol present, of˜1.16×10 ⁻⁵/S (k_(on)=0.8/s/μM) and 3.3×10⁻⁶/S(k_(on)=1.9/s/μM) from arabidopis HPPD.

In, variants of the above methods, off rates are measured either in thepresence or absence of˜25%v/v glycerol. In the presence of glycerolvalues obtained are generally˜1.5-3× slower than in its absence althoughsometimes the change is outside this range or, even, in the otherdirection. Further examples of data obtained are depicted in thefollowing Table 3. TABLE 3 Dissociation rate constants, (k_(off) values)governing dissociation of various inhibitors from complexes with variousHPPD enzymes. Each line represents data from a separate singleexperimental set. Arabidopsis P. fluorescens HPPD. Wheat HPPD HPPDk_(off) (s⁻¹) − k_(off) (s⁻¹) − k_(off) (s⁻¹) − glycerol glycerolglycerol Inhibitor (k_(off) (s⁻¹) + (k_(off) (s⁻¹) + (k_(off) (s⁻¹) +Structure glycerol) glycerol) glycerol) I 3.3 × 10⁻⁶ 1.0 × 10⁻³ 2 × 10⁻⁶(2.0 × 10⁻⁶) (3.8 × 10⁻⁴) (8 × 10⁻⁶) II 1.16 × 10⁻⁵  2.5 × 10⁻⁴ — 8.6 ×10⁻⁶ 3.5 × 10⁻⁴ 5 × 10⁻⁵ III 1.1 × 10⁻⁵ >2.0 × 10⁻⁴   IV (1.6 × 10⁻⁶)(1.66 × 10⁻⁵)  (4.2 × 10⁻⁵)   8.3 × 10⁻⁶ 6.2 × 10⁻⁵ 1.8 × 10⁻⁴   V 1.25× 10⁻⁶  4.2 × 10⁻⁶ >2 × 10⁻⁴   (2.7 × 10⁻⁷) VI 2.0 × 10⁻⁶ 2.5 × 10⁻⁵ >4× 10⁻⁴   VII   1 × 10⁻⁶ 8.3 × 10⁻⁶ >3 × 10⁻⁴  

Thus it can be seen, inter alia, that, according to the method,polynucleotides comprising a region which encodes an HPPD enzyme arescreened via a process of expression and testing in vitro in respect ofHPPD enzyme/HPPD inhibitor dissociation rates (k_(off) values). It canbe further seen from the above example that a polynucleotide comprisinga region which encodes wheat HPPD is selected as one which encodes aninhibitor-resistant HPPD because it is found that the ratio(k_(off)I/k_(off)3) of the value of koff for the complex of theexpressed wheat HPPD with structure I (a compound selected fromFormula 1) to that for the complex formed with structure IV (a compoundselected from Formula 3) is 22.9 or 16.13 which is>>2.5 fold more thanthe likewise derived ratio of 1.25 or 0.38 observed in respect ofdissociation of the same pair of inhibitors from Arabidopsis controlenzyme. As can be seen, the screening and comparison could equally aswell have been made, for example, in respect of structure II (a compoundselected from Formula 2) and structure VII (a compound selected fromformula 4) with the same result that a polynucleotide comprising aregion which encodes wheat HPPD is selected. In this case, the ratio,k_(off)2/k_(off)4 in respect of wheat HPPD is 30 or 42 which, againis>2.5 fold more than the equivalent ratio of 11 or 8.6 in respect ofthe Arabidopsis control enzyme. Alternatively, a polynucleotidecomprising a region which encodes wheat HPPD is screened and selected onthe basis that it encodes an HPPD-inhibitor resistant HPPD enzyme ableto form a complex in water at pH 7.0 and at 25 C with a herbicidal HPPDinhibitor, in this case structure I or structure II, wherein thedissociation of the said complex is governed by a rate constant(k_(off)) in the range from 4×10⁻⁵ to 2×10⁻³ s⁻¹ (in this case, 3.8×10⁻⁴or 1.0×10⁻³and 2.5×10⁻⁴/s or 3.5×10⁻⁴, respectively) and wherein theselected HPPD-inhibitor has at least a quarter of the herbicidalactivity of mesotrione versus dicot plants (this being true of structureII as, obviously, of mesotrione itself).

Alternatively, the example illustrates that, according to the method, apolynucleotide comprising a region which encodes Pseudomonas HPPD isselected as one which encodes an inhibitor-resistant HPPD because it isfound that the ratio (k_(off)4/k_(off)1) of the value of k_(off) for thecomplex of the expressed Pseudomonas HPPD with structure VI (a compoundselected from formula 4) to that for the complex formed with structure I(a compound selected from formula 1) is>21.7 or>100 which is>>2.5 foldmore than the likewise derived ratio of 0.6 observed in respect ofdissociation of the same pair of inhibitors from Arabidopsis controlenzyme. As can be seen, the screening and comparison could equally aswell have been made, for example, in respect of structure IV (a compoundselected from formula 3) and structure I (a compound selected fromformula 1) with the same result that a polynucleotide comprising aregion which encodes Pseudomonas HPPD is selected. In this case, theratio, k_(off)3/k_(off)1 in respect of Pseudomonas HPPD is 5.25 or 90which, again is >2.5 fold more than the equivalent ratio of 0.8 or 2.5in respect of the Arabidopsis control enzyme. Alternatively, apolynucleotide comprising a region which encodes Pseudomonas HPPD isscreened and selected on the basis that it encodes an HPPD-inhibitorresistant HPPD enzyme able to form a complex in water at pH 7.0 and at25 C with a herbicidal HPPD inhibitor, in this case structure IV orstructure VI, wherein the dissociation of the said complex is governedby a rate constant (k_(off)) in the range from 4×10⁻⁵ to 2×10⁻³ s⁻¹ (inthis case, 4.2×10⁻⁵ or 1.8×10⁻⁴ and>2.0×10⁻⁴/s, respectively) andwherein the selected HPPD-inhibitor has at least a quarter of the isherbicidal activity of mesotrione versus dicot plants (this being trueof both structure IV and VI).

EXAMPLE 11 In Vitro Screening and Selection of PolynucleotidesComprising Regions which Encode HPPD Enzymes Based Upon Measurement ofthe Relative and Absolute Values of the Dissociation Constants (KdValues) of Enzyme/Inhibitor Complexes

Qualitative measurements of the differences in Kd values in respect ofdifferent HPPD inhibitors are obtained by pre-incubating enzyme withinhibitor and, then, subsequently, measuring the % inhibition. Forexample HPPD is isolated from maize seedlings, part purified and assayedby similar methods to those described by Schulz et al 1993 (FEBS LETS.318, 162-166) and by Secor (1994) in Plant Physiol. 106, 1429-1433.Assays are run for 30 minutes and started with addition of radiolabelledhydroxyphenylpyruvate (final concentration 0.1-0.2 mM) following a 20-30min period over which inhibitor is pre-incubated with the part-purifiedenzyme. The following levels of inhibition (relative to controls) areobserved with the following doses of herbicide versus maize HPPDmesotrione sulcotrione structure IV   1 nM <17% — 44%  10 nM   44%  8%78%  100 nM   51% 51% 92% 1000 nM >75% 83% 93%

Using the same assay method but using HPPD from Arabidopsis (obtainedfrom E.coli cells transformed to express the Arabidopsis HPPD andprepared as an E.coli extract similar to the methods described by Garciaet al in Plant Physiol (1999) 119, 1507-1516) the following levels ofinhibition (relative to controls) are observed with the following dosesof herbicide versus Arabidopsis HPPD. mesotrione sulcotrione structureIV   1 nM  42%  57% 20%  10 nM  95%  96% 90%  100 nM 100% 100% 95% 1000nM 100% 100% 95%

The experiment indicates that structure IV is a similar or somewhat lesspotent inhibitor of Arabidopsis HPPD than mesotrione and sulcotrione(which appear˜10 and 100 fold less active against the maize enzyme thanstructure IV). These data demonstrate that some substituted 1,3cyclohexane dione herbicides such as sulcotrione and mesotrione have, asat least a part of the basis of their observed selectivity, a tendencyto inhibit HPPD from monocotyledonous plants less strongly than thatfrom dicotyledenous plants. In order to obtain quantitative measurementsof absolute and relative Kd values methods are used as described below.

Crude Extracts of recombinant E.coli strains expressing, in the onecase, a control HPPD (from Arabidopsis) and, in the other, one or moretest HPPD sequences are prepared as described in the preceding example.The titre of active sites and enzyme activity are also defined andmeasured as described in the preceding examples. The dissociation rates(off rates) of inhibitors selected from formula 1 and/or 2 and/or 3and/or 4 from the complexes that they form with test and control HPPDenzymes are suitably measured as described in the preceding examples. Kdvalues are suitably calculated from the ratio k_(off)/k_(on). Invariants of the method, on rates and off rates are both measured in thepresence of˜25% v/v glycerol or, both are measured in its absence.Generally glycerol appears to slow on and off rates to about the sameextent and therefore Kd values often do not appear to vary withglycerol. On some occasions though Kd is glycerol dependent. Rateconstants (k_(on) values) governing the rate of binding of inhibitors toHPPD enzymes are suitably measure in a number of ways.

Firstly HPLC assays monitoring the formation of HGA at various times arerun as described the preceding example 9. Even in the absence of addedinhibitors, progress curves are not linear reflecting progressiveinactivation of the enzyme under assay conditions. At increasingconcentrations of inhibitor the curvature becomes much more marked. Itis possible to fit both control and inhibited curves to a simpleexponential decay in the amount of active enzyme from the startingconcentration toward a final level of zero (i.e. a decline in theconcentration of active enzyme governed by a process where E(t)=E(o).e^(−kt). Thus, apparent rate constants (k′) at a range ofdifferent concentrations of inhibitor are derived by subtracting thecontrol rate constant fitted to the progress curve in the absence ofinhibitor from the observed rate. Estimates of true rate constants(k_(on)) are then derived by multiplying apparent rate constants, k′, by1/(1+S/Km) where S is the concentration of HPP in the assay. Given theneed for curve subtraction and to know the value of Km this is probablythe least accurate of the various methods of determining k_(on).Nevertheless it is valuable since it provides a direct test of thepresumption, implicit in the calculation, that inhibitor binding can beadequately described by a simple E+I ← → EI binding process rather thana more complex scheme involving the initial rapid formation of a rapidlydissociable enzyme inhibitor complexes which then slowly isomerizes to amore tightly inhibited form. In the latter case, rather than zeroinhibition at near zero time, a significant level of initial inhibitionis seen (Schloss, J. V.(1989) in “Target sites of Herbicide Action”(Boger, P.and Sandmann G. eds) CRC Press Boca). An example experiment isillustrated in FIG. 3.

In FIG. 3 assays containing 100 μM HPP substrate were started withaddition of wheat HPPD to a final concentration of˜19 nM and stopped atthe times indicated. The upper progress curve is with no inhibitorpresent, the middle with 2.5 μM and the lower with 10 μM of structure IIpresent; these curves are fit to an initial rate of 0.35 μM/s (i.e.there is no significant initial inhibition) with observed first orderdecay rate constants of 0.016, 0.029 and 0.06/s, respectively. Assuminga Km value of 10 μM, the value of k_(on) is therefore estimated asbetween 48000 and 57200 s⁻¹M⁻¹.

In a more direct assay-based method for measuring k_(on) values, testHPPD enzyme is reacted with inhibitor for a range of times and then theinhibition reaction is effectively stopped by addition of a highconcentration of the (competitive) substrate HPP. At the same time aseffectively freezing further inhibition this also starts the assay whichindicates how far inhibition had proceeded during the period before theHPP was added. The following example illustrates the method. Wheat HPPDis diluted to a concentration of 0.465 μM in 50 mM Bis-Tris propanebuffer at pH 7.0 containing 25 mM sodium ascorbate and 3.9 mg/l ofbovine catalase (Sigma C3155-50,000 units/mg). 5 μl of diluted HPPD isadded to, alternatively, 10 μl of 50 mM BTP buffer at pH 7.0 (control),10 μl of 50 mM BTP buffer at pH 7.0 containing 0.5 μM structure VI or 10μl of 50 mM BTP buffer at pH 7.0 containing 2.0 μM structure VI. Thereactions are left to run for alternative times of 0 (‘pre-stopped’assay), 10, 30, 50 or 70 s at 25 C before addition of 100 μl of 150 μMHPP. After addition of HPP, assays are run for 40 s before stopping withaddition of 20 μl of 25% perchloric acid and analysis by HPLC. In thetimed ‘pre-reactions’ between enzyme and inhibitor the concentration ofenzyme is 0.155 μM that of inhibitor, alternatively, 0, 0.33 μM and 1.33μM. Note that, because the initial, fast, reaction of inhibitors withHPPD which results in complete loss of activity is with only half thesites ultimately measurable by binding stoichiometry, the relevantenzyme concentration here for simulation and for calculation of rateconstants is half the enzyme concentration as measured in long-termtitration binding studies as described above. In the assays run for 40s, the maximum final concentration of inhibitor is 0.174 μM. It isconfirmed through experiments such as that described for FIG. 3 that,especially in the presence of 130 μM HPP, this is far too little tocause any detectable progressive inhibition during the course of the 40s assay itself and, thus, that all the inhibition observed is due toinhibitor binding to enzyme occurring during the timed pre-reaction inthe absence of substrate. The data obtained are fit to a model E+I→EIreaction wherein ‘activity’ is equivalent to ‘active enzyme’ and thedecline in activity mirrors the decline in the species ‘E’ afteraddition of inhibitor. Given the relatively very low values of off ratesit can be assumed that the inhibition reaction is effectivelyirreversible under the reactions conditions (it makes no significantdifference if the reaction is modelled as a reversible one and the knownlow off rates are included in the fit). The results from the experimentand fit to the data are illustrated in FIG. 4. The upper graphrepresents the rate of enzyme in activation at 0.33 μM structure VI, thelower graph, the rate at 1.33 μM. Both curves are fit to a rate constant(k_(on)) value of 70,000 M⁻¹ s⁻¹.

The measurements of k_(off) are based on physical rather thanassay-based measurements. Similarly, k_(on) rates can also be measuredby a direct physical method, in this case the use of radiolabelledinhibitor and physical separation of protein bound from free inhibitor.It is useful to obtain the correlation between physical binding andassay-based methods because, for example, it can show, especially wherephysical binding indicates, initially, only ‘half of the sites’ bindingthat this, nevertheless, occurs contemporaneously with the loss of allof the enzyme activity. The rates of binding determined on the basis ofmeasurement of the amount of radiolabelled inhibitor bound after varioustimes of reaction are found to correlate very well with measurementsbased upon assay-based measurements of the rate of decline of enzymeactivity. Furthermore it is also found that measurements of k_(off)based upon exchange studies as described elsewhere herein yield similarresults independently of whether or not the initial binding reaction toform enzyme inhibitor complex is stopped after 10 s (such that thereaction is only partly complete) or after 10 h, confirming that the onrates and off rates which are measured relate to the same species ofenzyme/inhibitor complex (rather, for example, than there being aninitial weak inhibited complex for which we measure on rates whichisomerises slowly to a tighter-bound complex for which we measure offrates) and thus, the two values can be validly combined to yield valuesof Kd.

An illustrative example of an experiment to measure the on rate ofmesotrione binding to Arabidopsis HPPD follows. A series of eppendorfcentrifuge tubes are set up at 25 C containing ¹⁴C mesotrione in 50 mMBis-Tris propane buffer at pH 7.0 containing 25 mM sodium ascorbate, 25%v/v glycerol and 3.0 mg/l of bovine catalase (Sigma C3155-50,000units/mg). Reactions are started by addition of Arabidopsis HPPD suchthat the final concentrations of Arabidopsis HPPD and ¹⁴C mesotrioneare, ˜0.30 μM and 0.347 μM, respectively, mixed and rapidly stoppedafter various time intervals by addition of a large excess of unlabelledmesotrione to a final concentration of 170 μM. After stopping samplesare quickly separated into protein-bound and protein-free fractions byrapid Gel filtration down a NAP5 Pharmacia column equilibrated in 50 mMBTP at pH 7 containing 0.1 M KCl and the radioactivity in the twofractions measured by liquid scintillation counting. Results obtainedand the fitting of data are illustrated in FIG. 5.

The data of FIG. 5 are fit to a rate constant, k_(on) value of 125000M⁻¹ s⁻¹ with only half of the Arabidopsis sites binding mesotrione.There is a subsequent much slower reaction not shown (fit to a rateconstant of˜1000 M⁻¹ s⁻¹) in which mesotrione binds to the remaininginhibitor site. Inhibitor/HPPD combinations are found to vary in whetheror not only half the sites are bound initially. In either case it isonly the initial rate, as depicted in FIG. 5, which is taken to be thevalue of k_(on). In a similar experiment to FIG. 5 but in the absence ofglycerol the value of k_(on) is found to be 190000 M⁻¹ s⁻¹. This valueis indistinguishable from the value of˜250000 M⁻¹ s⁻¹ found using assaybased measurements of the rate of activity loss. Similar bindingexperiments indicate, for example, a value of 100000 M⁻¹ s⁻¹ for therate constant, k_(on) governing the association of structure IV withArabidopsis HPPD in the presence of glycerol. The, above-described,methods for the measurement of ko, and k_(off) values allow calculationof Kd values, some of which are illustrated in Table 4. TABLE 4Dissociation constants, (Kd values) governing dissociation of variousinhibitors from complexes with various HPPD enzymes Arabidopsis Wheat P.fluorescens HPPD. HPPD HPPD Kd (pM) Kd (pM) Kd(pM) (value (value (valueInhibitor obtained + obtained + obtained + Structure glycerol) glycerol)glycerol) I 14 7407   114 (21) (6333)   (200) II 110  6727  2174 III IV46 885 12200  (17) (596) (1100) V  4  11 >1500   VI 25 450 >20000    VII32 175

Thus it can be seen, inter alia, that, according to the method,polynucleotides comprising a region which encodes an HPPD enzyme arescreened via a process of expression and testing in vitro in respect ofHPPD enzyme/HPPD inhibitor dissociation constants (Kd values). It can befurther seen from the above example that a polynucleotide comprising aregion which encodes wheat HPPD is selected as one which encodes aninhibitor-resistant HPPD because it is found that the ratio (Kd1/Kd3) ofthe value of Kd for the complex of the expressed wheat HPPD withstructure I (a compound selected from formula 1) to that for the complexformed with structure IV (a compound selected from formula 3) is 83.7 or14.3 which is>>2.5 fold more than the likewise derived ratio of 0.3 or1.1 observed in respect of dissociation of the same pair of inhibitorsfrom Arabidopsis control enzyme under the same conditions. As can beseen, the screening and comparison could equally as well have been made,for example, in respect of structure II (a compound selected fromformula 2) and structure VII (a compound selected from formula 4) withthe same result that a polynucleotide comprising a region which encodeswheat HPPD is selected. In this case, the ratio, Kd2/Kd4 in respect ofwheat HPPD is 38 which, again is>2.5 fold more than the equivalent ratioof 3.4 in respect of the Arabidopsis control enzyme. Alternatively, apolynucleotide comprising a region which encodes wheat HPPD is screenedand selected on the basis that it encodes an HPPD-inhibitor resistantHPPD enzyme able to form a complex in water at pH 7.0 and at 25 C with aherbicidal HPPD inhibitor, in this case structure I or structure II,wherein the dissociation of the said complex is governed by adissociation constant (Kd) in the range from 1.0 to 30 nM (in this case,˜6.5-7.5 nM) and wherein the selected HPPD-inhibitor has at least aquarter of the herbicidal activity of mesotrione versus dicot plants(this being true of structure II as, obviously, of mesotrione itself).

Alternatively, the example illustrates that, according to the method, apolynucleotide comprising a region which encodes Pseudomonas HPPD isselected as one which encodes an inhibitor-resistant HPPD because it isfound that the ratio (Kd3/Kd 1) of the value of Kd for the complex ofthe expressed Pseudomonas HPPD with structure IV (a compound selectedfrom formula 3) to that for the complex formed with structure I (acompound selected from formula 1) is107 which is>2.5 fold more than thelikewise derived ratio of 3.3 observed in respect of dissociation of thesame pair of inhibitors from Arabidopsis control enzyme. As can be seen,the screening and comparison could equally as well have been made, forexample, in respect of structure V (a compound selected from formula 4)and structure I (a compound selected from formula 1) with the sameresult that a polynucleotide comprising a region which encodesPseudomonas HPPD is selected. In this case, the ratio, Kd4/Kd1 inrespect of Pseudomonas HPPD is>4.3 which, again is>2.5 fold more thanthe equivalent ratio of 0.28 in respect of the Arabidopsis controlenzyme. Alternatively, a polynucleotide comprising a region whichencodes Pseudomonas HPPD is screened and selected on the basis that itencodes an HPPD-inhibitor resistant HPPD enzyme able to form a complexin water at pH 7.0 and at 25 C with a herbicidal HPPD inhibitor, in thiscase structure IV or structure VI, wherein the dissociation of the saidcomplex is governed by a dissociation constant (Kd) in the range from 1to 30 nM (in this case, for example, 12.2 nM) and wherein the selectedHPPD-inhibitor has at least a quarter of the herbicidal activity ofmesotrione versus dicot plants (this being true of both structure IV andVI).

EXAMPLE 12 Production of Stably-Transformed Morphologically NormalFertile Soyabean Plants which Comprise a DNA Region Encoding an Avenasativa HPPD Enzyme and which are Resistant to HPPD-Inhibitor Herbicides

Suitable polynucleotides for plant transformation comprising a gene forexpression of Avena sativa HPPD are described, for example, in theprevious examples. Optionally, the HPPD gene itself can provide themeans of selection and identification of transgenic tissue. Optionallythe gene for expression of Avena sativa HPPD can be present in thepolynucleotide alongside other sequences which provide additional meansof selection/identification of transformed tissue including, forexample, genes which provide resistance to kanamycin, hygromycin,phosphinothricin or glyphosate. Alternatively these selectable markersequences may be present on separate polynucleotides and a process of,for example, transformation by co-bombardment and co-selection is used.Alternatively, rather than a selectable marker gene a scorable markergene such as GUS may be used to identify transformed tissue. Soybeanplant material can be suitably transformed and fertile plantsregenerated by many methods which are well known to the skilled man. Forexample, fertile morphologically normal transgenic soybean plants may beobtained by 1) production of somatic embryogenic tissue from e.g.immature cotyledon, hypocotyl or other suitable tissue 2) transformationby particle bombardment or infection with Agrobacterium and 3)regeneration of plants.

Alternatively such soybean plants may be obtained by infection of budsand/or flower tissues with Agrobacterium by vacuum infiltration andselection of transgenic seed and/or plants grown from rescued embryos.In one example, as described in U.S. Pat. No. 5,024,944, cotyledontissue is excised from immature embryos of soybean, preferably with theembryonic axis removed, and cultured on hormone-containing medium so asto form somatic embryogenic plant material. This material is transformedusing, for example, direct DNA methods, DNA coated microprojectilebombardment or infection with Agrobacterium, cultured on a suitableselection medium and regenerated, optionally also in the continuedpresence of selecting agent, into fertile transgenic soybean plants.Selection agents may be antibiotics such as kanamycin, hygromycin orherbicides such as phosphonothricin or glyphosate or, alternatively,selection may be based upon expression of a visualisable marker genesuch as GUS. Alternatively target tissues for transformation comprisemeristematic rather than somaclonal embryogenic tissue or, optionally,is flower or flower-forming tissue.

In one example, constructs are transformed into regenerable embryogenicsoybean tissues using either biolistic type approaches (e.g Santarem ER,Finer, J. J (1999) Transformation of soyabean (Glycine max (L.) Merrill)using proliferative embryogenic tissue maintained on a semi-solidmedium. In vitro Cellular and Developmental Biology-Plant 35, 451-455;U.S. Pat. No. 5,503,998, U.S. Pat. No. 5,830,728) or via infection withAgrobacterium (e.g.,U.S. Pat. No. 5,024,944, U.S. Pat. No. 5,959,179).Regenerable embryogenic soybean tissues are derived, for example, fromthe cotyledons of immature embryos.

Proliferative embryogenic tissue can, for example, be maintained on asemi-solid medium. Such tissue, is, for example obtained in thefollowing way. Immature zygotic embryos which are 3-4 mm long areisolated from pods of, for example, Glycine max (L.) Merrill, 2-3 weeksafter flower formation. Pods can be checked for the presence of embryosof the correct length and maturity by ‘backlighting’. Pods are thensterilized. Immature embryos are removed and the axis removed from each.Immature embryos are then plated on ‘D40-Lite’ semi-solid (0.2% gelrite)MS salts medium at pH 7.0 containing B5 vitamins, 3% sucrose and 40 mg/l2,4-D for 3-4 weeks. For proliferation of embryos the material is thentransferred to D20′ MS salts medium at pH 5.7 containing B5 vitamins, 3%sucrose, 20 mg/l 2,4-D and 0.2% Gelrite. Material with bright greenglobular proliferative embryos is selected and subcultured every 2-3weeks.

For bombardment, 20-25 clumps/plate of tissue are selected (subcultured4-5 days prior to bombardment) and arranged in the centre of the dishcontaining D20 medium. The tissue is dried for 15 min by uncovering for15 minutes under a sterile hood. Gold particles coated in DNA construct(coated, for example, using methods described in the references above)are twice bombarded into the tissue on D20 medium using any one of alarge number of commercially available guns. By way of further example aPDS1000 particle gun is used. Particles may be prepared and coated withDNA in a similar manner to that described by Klein et al 1987, Nature,327, 70-73.

Alternatively, for example, 60 mg of gold or tungsten particles (˜1.0μm) in a microcentrifuge tube are washed repeatedly in HPLC-gradeethanol and then, repeatedly, in sterile water. The particles areresuspended in 1 ml of sterile water and dispensed into 50 μl aliquotsin microcentrifuge tubes. Gold particles are stored at 4 C, tungstenparticles at—20 C. 3 mg of DNA are added to each aliquot of (defrosted)particles and the tubes are vortexed at top speed. Whilst maintainingnear continuous vortexing, 50 μl of 2.5M CaCl₂ and 20 μl of 0.1Mspermidine is added. After 10 minutes of further vortexing, samples arecentrifuged for 5 seconds in an eppendorf microcentrifuge, thesupernatant is drawn off and the particles washed in successiveadditions of HPLC-grade ethanol. The particles are thoroughlyresuspended in 60 μl of ethanol and then dispensed in 10 μl aliquotsonto the surface of each macrocarrier to be used in the PDS1000 particlegun. Components of the PDS1000 particle gun are surface sterilised byimmersion in 70% ethanol and air-drying. Target plates prepared, asdescribed above, with tissue arranged into an 2.5 cm disc are placed 6cm from the stopping screen. Suitably chosen rupture discs are then usedfor bombardment.

One week after bombardment, all tissue clumps are transferred onto D20medium, buffered to pH 5.7, containing a suitable selectiveconcentration of selecting agent (for example glyphosate between 0.05and 10 mM in the case that glyphosate be used for selection and that aresistant EPSPS or GOX encoding gene is either present on the sametransforming DNA as the gene expressing Avena sativa HPPD or, otherwise,is present in co-bombarded DNA). After an additional 3-4 weeks alltissue is transferred to fresh D20 medium containing a suitableincreased concentration of selecting agent. After a further 3-4 weeks,living tissue is selected and subcultured on every 3-4 weeks in similarD20 medium containing selection agent. In the case that some otherselectable marker than glyphosate is present then selections may be madeas appropriate (e.g., using increasing concentrations of hygromycin).Alternatively, all selections are made using HPPD inhibitor herbicides.Growing sections are thus maintained and, given enough tissue, may beanalysed by PCR to confirm that they are transgenic for the desired DNA.

In order to develop and mature embryos, tissue clumps are placed onto M6medium which comprises MS salts at pH 5.7 containing B5 vitamins, 6%maltose and 0.2% gelrite. 6-9 clumps are placed in a tall dish at 23° C.After 3-4 weeks, embryos elongate and can be separated and transferredto another round of incubation on M6 medium. After 4-6 weeks, embryosare cream-coloured and ready for desiccation. 9 such cream-colouredembryos are placed in a dry Petri dish, sealed with parafilm and placedonto a shelf for 2-3 days. Embryos should be somewhat flaccid and not“crispy-crunchy”.

Dessicated embryos can be germinated by plating onto OMS (growthregulator-free MS medium). Following germination which normally occurswithin a week plants are transferred to larger boxes and, once there issufficient root and shoot formation, thence to soil. To prevent fungalcontamination it is advisable to wash OMS from the roots with distilledwater. Plants may be kept and grown under high humidity and, initially,under 24 hour lighting. Plants may be grown until about 2 feet 15 tallunder 24 hour lighting and then encouraged to flower and form podsthrough a shift to a 16 hour lighting regime. Seeds are collected andprogeny grown on, crossed and backcrossed into order to move thetransgenes into the desired plant background using the normal methods ofplant breeding. Plants are routinely analysed for the presence andexpression of transgenes using the normal methods of molecular biologyincluding analysis by PCR, Southern, Western, ELISA and enzyme assaytechniques.

EXAMPLE 13 Production of Stably-Transformed Morphologically NormalFertile Corn Plants which Comprise a DNA Region Encoding an Avena sativaHPPD Enzyme and which are Resistant to HPPD-Inhibitor Herbicides

Constructs for corn transformation preferably have the DNA sequenceencoding Avena sativa HPPD under operable expression control of themaize polyubiquitin promoter and also include a suitable terminatorsequence such as that from the 3′ end of the Nos gene. Optionally thisDNA sequence also comprises a sequence which provide an additional meansof selection/identification of transformed tissue including, forexample, genes which provide resistance to kanamycin, butafenacil,hygromycin, phosphinothricin , glyphosate, or postive mannose selection.Alternatively these selectable marker sequences may be present onseparate polynucleotides and a process of, for example, transformationby co-bombardment and co-selection is used. Alternatively, rather than aselectable marker gene a scorable marker gene such as GUS may be used toidentify transformed tissue. The DNA sequence may be delivered to corntarget tissue using many methods which are well known in the artincluding (i) via placement within the left and right borders of a T-DNAsequence and infection with Agrobacterium (ii) as a DNA coating onmicroprojectiles and bombardment (iii) as a coating on silicon carbidewhiskers or iv) by direct DNA delivery methods.

EXAMPLE 14 Transformation of Corn using Agrobacterium

For example, DNA comprising the HPPD sequence is ligated into a positionwithin the cloning site located between the right and left T-DNA bordersof similarly restricted pSB 11. The construction of plasmid pSB 11 andthe construction of its parent, pSB21, is described by Komari et al(1996, Plant J. 10: 165-174). The T-DNA region comprising the HPPDsequence is then integrated into the superbinary pSB1 vector.(Saito etal EP 672 752 Al) by a process of homologous recombination. To achievethis the psB11 comprising the HPPD sequence is transformed into E. colistrain HB101 which is then, according to the triple cross method ofDitta et al (1980, Proc. Natl. Acad. Sci. USA 77: 7347-7351), mated withan Agrobacterium LBA4404 harbouring pSB1 to create the transformedstrain of Agrobacterium, LBA4404 (pSB1-HPPD) in which the presence ofthe cointegrate plasmid pSB1-HPPD is selected for on the basis ofresistance to spectinomycin. The identity of pSB1-HPPD is also confirmedon the basis of Sal 1 restriction analysis.

Alternatively, using similar methods to those described above, a similarfragment of HPPD sequence is homologously recombined into a positionbetween the right and left borders of the superbinary vector pTOK162(FIG. 1 in U.S. Pat. No. 5,591,616) to generate a similar set ofcointegrate plasmids selected for in Agrobacterium on the basis ofcombined resistance to kanamycin and spectinomycin.

Agrobacterium strain LBA4404 which has a helper plasmid PAL4404 (havinga complete vir region) is available from the American Type CultureCollection (ATCC 37349). An alternative useful strain is AgrobacteriumEHA101 (1986, Hood et al, J. Bacteriol., 168(3): 1283-1290) which has ahelper plasmid having the vir region from the strongly virulent strainAgrobacterium tumefaciens A281.

Agrobacterium strains LBA4404(pSB1-HPPD) etc are each streaked ontoplates containing, for example, ‘PHI-L’ solid medium and cultured at 28C in the dark for 3 to 10 d. PHI-L medium is as described on page 26(Example 4) of WO 98/32326. Alternatively the Agrobacterium are culturedfor 3-10 d on a plate containing YP medium (5 g/l yeast extract, 10 g/lpeptone, 5 g/l NaCl, 15 g/l agar at pH 6.8) as described by Ishida et al(1996, Nature Biotechnology, 14, 745-750) or, alternatively, asdescribed by Hei et al in US 5591616 (AB medium (Drlica and Kado, 1974;Proc. Natl. Acad. Sci. USA 71:3677-3681)) but, in each case, modified toprovide the appropriate antibiotic selection (e.g. containing 50 jig/mlspectinomycin in the case of Agrobacterium strain LBA4404(pSB1-HPPD)etc. or containing both 50 μg/ml spectinomycin and 50 μg/ml kanamycin inthe case that Agrobacterium containing a pTOK 162-derived superbinaryvector is used).

Plates of Agrobacterium made as described above are stored at 4 C andused within a month of preparation. Suspensions of Agrobacterium fortransformation of plant material are prepared in a similar manner todescribed in U.S. Pat. No. 5,591,616. (Using good microbiologicalpractice to avoid contamination of aseptic cultures) 3×5 mm loopfuls ofAgrobacterium are removed from plates, transferred and suspended in 5 mlof sterile AA liquid medium in a 14 ml Falcon tube. Alternatively,suspensions of Agrobacterium for transformation of plant material areprepared in a similar manner to described in WO 98/32326. 3×5 mmloopfuls of Agrobacterium are removed from plates, transferred andsuspended in 5 ml of the sterile PHI-A basic medium as described inExample 4 on page 26 of WO 98/32326 or, alternatively, suspended in 5 mlof the sterile PHI-I combined medium also described in Example 4 on page26 of WO 98/32326. Alternatively, suspensions of Agrobacterium fortransformation of plant material are prepared in a similar manner todescribed by Ishida et al (1996) Nature Biotechnology, 14, 745-750.However produced, the suspension of Agrobacterium is vortexed to make aneven suspension and the cell population adjusted to between 0.5×10⁹ and2×10⁹ cfu/ml (preferably the lower). 1×10⁹ cfu/ml corresponds to an OD(1 cm) of˜0.72 at 550 nm. Agrobacterium suspensions are aliquoted into 1ml lots in sterile 2 ml microcentrifuge tubes and used as soon aspossible

Suitable maize lines for transformation include but are not restrictedto, A188, F1 P3732, F1 (A188×B73Ht), F1 (B73Ht×A188), F1 (A188×BMS).Suitable maize lines also include a variety of A 188×inbred crosses (e.gPHJ90×A 188, PHN46×A188, PHPP8×A188 in table 8 of WO98/32326) as well aselite inbreds from different heterotic groups (e.g PHN46, PHP28 andPHJ90 in table 9 of WO98/32326).

In a particular example immature embryos are produced from “Hi-II” corn.“Hi-II” is a hybrid between inbreds (Al 88 x B73) generated byreciprocal crosses between Hi-II parent A and Hi-II parent B availablefrom the Maize Genetic Cooperation Stock Center, University of Illinoisat Champaign, Urbana, Ill.). Seeds, termed ‘Hi-II’ seeds obtained fromthese crosses are planted out in a greenhouse or field. The resultingHi-II plants are self or cross-pollinated with sister plantsTransformation of immature embryos of corn is carried out by contactingthe immature embryos with the suitable recombinant strains ofAgrobacterium described above. An immature embryo means the embryo of animmature seed which is in the stage of maturing following pollination.Immature embryos are an intact tissue that is capable of cell divisionto give rise to callus cells that can then differentiate to produce thetissues and organs of a whole plant. Preferred material fortransformation also includes the scutella of embryos which is alsocapable of inducing dedifferentiated calli with the ability toregenerate normal fertile plants having been initially transformed.Preferred material for transformation thus also includes callus derivedfrom such dedifferentiated immature zygotic embryos or scutella.

Immature corn embryos are isolated aseptically from developing ears asdescribed by Green and Phillips (1976, Crop. Sci. 15: 417-421) or,alternatively, by the methods of Neuffer et al (1982, “Growing Maize forgenetic purposes” in Maize for biological research, W. F. Sheridan ed.,University Press, University of North Dakota, Grand Forks, N.Dak., USA).For example, immature corn embryos between 1-2 mm (preferably 1-1.2 mm)long are aseptically isolated from female spikes at 9-12 (preferably 11)d after pollination using a sterile spatula. Typically ears are surfacesterilised with 2.63% sodium hypochlorite for 20 min before washing withsterile deionized water and aseptic removal of immature embryos.Immature embryos (preferably˜100 in number) are dropped directly into a2 ml microcentrifuge tube containing about 2 ml of the same medium asused for preparing the suspension of Agrobacterium (the alternatives forwhich are described above). The cap of the tube is closed and thecontents mixed by vortexing for a few seconds. The medium is decantedoff, 2 ml of fresh medium are added and vortexing is repeated. All ofthe medium is then drawn off to leave the washed immature embryos at thebottom of the tube.

Having prepared the immature maize embryos the next phase of theprocess, the infection step, is to contact them with the transformedstrain of Agrobacterium. In one example of this process, the infectionstep takes place in a liquid medium which includes the major inorganicsalts and vitamins of N6 medium (1987, Chu C. C. Proc.

Symp. Plant Tissue Culture, Science Press Peking. Pp 43-50) as describedin example 4 of WO 98/32326. For example, as described in WO 98/32326,1.0 ml of suspension of Agrobacterium, prepared as described above inPHI-A medium is added to the embryos in the microcentrifuge tube andvortexed for about 30s. Alternatively, 1.0 ml of suspension ofAgrobacterium prepared, also as described above, in either PHI-I mediumor in LS-inf medium is added. After standing for 5 minutes thesuspension of Agrobacterium and embryos is poured out into a Petri platecontaining either 1) PHI-B medium or 2) PHI-J medium or 3) LS-AS mediumaccording to whether the original suspension of Agrobacterium had beenprepared in PHI-A medium, PHI-I medium or LS-inf medium, respectively.The Agrobacterium suspension is drawn off using a Pasteur pipette, theembryos manipulated so that they sit axis-side downwards onto themedium, the plate sealed with parafilm and incubated in the dark at23-25 C for 3 days of cocultivation.

Following the preparation of immature embryos, an alternative method ofachieving transformation is to infect them during and after a period ofdedifferentiation as described in U.S. Pat. No. 5,591,616. Immatureembryos are placed on LSD 1.5 solid medium containing LS inorganic saltsand vitamins along with 100 mg/ml casamino acids, 700 mg/l L-proline,100 mg/l myo-inositol, 1.5 mg/ml of 2,4-D, 20 g/l sucrose and 2.3 g/l ofgelrite. After 3 weeks at 25 C, calli originating from the scutella arecollected in a 2 ml microcentrifuge tube and immersed in 1 ml ofAgrobacterium suspension prepared, as described above, in AA medium.After standing for 5 minutes, the resultant calli are transferred to 2N6solid medium containing 100 JIM acetosyringone and incubated in the darkat 25 C for a 3 day period of cocultivation.

For the selection step, about 20 embryos are transferred onto each of anumber of fresh plates containing PHI-D (WO 98/32326) selection mediumor LSD 1.5 (U.S. Pat. No. 5,591,616) selection medium, sealed withparafilm and incubated in the dark at 28 C. Selection media, adjusted topH 5.8 with KOH, contain, depending upon the presence of selectablemarker genes, suitable concentrations of selecting agents. For example,an HPPD-inhibitor herbicide and/or, in the case that a gene encoding aresistant EPSPS enzyme be used, concentrations of between 0.1 mM and 20mM of tissue culture grade N-concentrations of (Phosphonomethyl)-glycine(Sigma P-9556) may be used. In the case that a resistantprotoporphyrinogen oxidaser gene be used, then butafenacil can be usedas selection agent. Alternatively selection media are designed to givepositive selection on mannose (Positech™ technology). Alternatively, inthe case that the starting material for selection are calli-derived fromimmature embryos as disclosed in WO 5591616 then such calli are washedwith sterilised water containing 250 mg/I cefotaxime before culturing onLSD 1.5 selection medium adjusted to provide suitable selection.

The embryos or clusters of cells that proliferate from the immatureembryos are transferred (if necessary using a sterile scalpel) to platescontaining fresh selection medium at 2 weekly intervals over a totalperiod of about 2 months. Selected calli are then bulked by continuedgrowth on the same medium until the diameter of the selected callusexceeds about 1.5 cm.

The selected calli are regenerated into normal fertile plants accordingto, for example, the methods described by Duncan et al (1985, Planta,165, 322-332) by Kamo et al (1985, Bot. Gaz. 146(3), 327-334) and/or byWest et al (1993, The Plant Cell, 5, 1361-1369) and/or by Shillito et al(1989) Bio/Technol. 7, 581-587. Optionally the regeneration medium mayalso be adjusted to provide for continued positive mannose selection orother selection.

For example, selected calli of diameter 1.5-2 cm are transferred toregeneration/maturation medium and incubated in the dark for about 1-3weeks to allow the somatic embryos to mature. A suitable regenerationmedium, is, for example, PHI-E medium (WO 98/32326) adjusted to pH 5.6with KOH, which may also, optionally, contain selection agent or beadjusted to provide for continued positive mannose selection.

The calli are then transferred to rooting/regeneration medium and grownat 25 C under either a schedule of 16 h daylight (270 mE m⁻² s⁻¹) and 8h of darkness or under continuous illumination ( 250 mE m⁻² s⁻¹) untilsuch a time as shoots and roots develop. Suitable rooting/regenerationmedia are either LSZ medium (optionally, including or not including,continued selection). Alternatively, selected calli are transferreddirectly to LSZ regeneration medium adjusted to pH 5.8 with KOH andcomprising LS major and minor inorganic salts (Linsmaier and Skoog,1965, Physiol. Plant 18,100-127), 0.5 mg/ml nicotinic acid, 0.5 mg/mlpyridoxine. HCl, 1.0 mg/ml thiamine. HCL, 700 mg/l L-proline, 100 mg/lmyo-inositol, 5 mg/ml of zeatin, 20 g/l sucrose, 0.5 g/l MES, 250 mg/Icefotaxime, 8 g/l purified agar (Sigma A-7049) or, optionally, suitablyadapted to provide continued selection (for example, on mannose, orcontaining an HPPD-inhibitor herbicide or glyphosate etc). After aperiod of incubation in the dark plates are subject to illumination(continuous or light/day as above)and plantlets regenerated.

Small plantlets are transferred to individual glass tubes containing,for example, either PHI-F medium or half strength LSF medium at pH 5.8comprising LS major salts (Linsmaier and Skoog, 1965, Physiol. Plant 18,100-127) at half strength, LS minor salts, 0.5 mg/ml nicotinic acid, 0.5mg/ml pyridoxine. HCl, 1.0 mg/ml thiamine. HCL, 100 mg/l myo-inositol,20 g/l sucrose, 0.5 g/l MES, 8 g/l purified agar (Sigma A-7049).andgrown on for about another week. Plantlets are then transferred to potsof soil, hardened off in a growth chamber (85% relative humidity, 600ppm CO₂ and 250 mE m⁻² s⁻¹) and grown to maturity in a soil mixture in agreenhouse.

The first (To) generation of plants obtained as above are selffertilised to obtain second generation (T1) seeds. Alternatively (andpreferably) the first generation of plants are reciprocally crossed withanother non-transgenic corn inbred tine in order to obtain secondgeneration seeds. The progeny of these crosses (T1) are then expected tosegregate 1:1 for the herbicide resistance trait. T1 seeds are sown,grown up in the glass house or field and the level of resistance,inheritance of resistance and segregation of resistance to selectedHPPD-inhibitor herbicides through this and subsequent generationsassessed by the observation of differential plant survival and the easyto score symptoms of bleaching and chlorosis following spray treatmentwith suitably formulated HPPD-inhibitor herbicides such as structure VI,isoxaflutole and structure II at a range of rates between 5 and 2000g/ha and at a range of growth stages between and including V2 and V8(or, alternatively, at 7-21 days post germination). These assessmentsare made relative to susceptible segregants and relative to similar,untransformed lines of corn which do not comprise genes of the presentor similar inventions capable of conferring resistance to glyphosate.Transgenic lines which exhibit high-level resistance to HPPD-inhibitorherbicides are selected and again selfed or backcrossed to anon-transgenic inbred.

At all stages in the above process tissue samples of transformed callus,plantlets, T0 and T1 plant material are optionally taken and analysedby 1) Southems and PCR in order to indicate the presence , copy numberand integrity of transgenes, 2) Northern (or similar) analysis in orderto measure expression of mRNA from transgenes, 3) quantitative Westernanalysis of SDS gels in order to measure expression levels of EPSPS and4) measurement of HPPD enzyme activity Such methods of analysis are wellknown in the art. Suitable methods to test for the presence, integrity,and expression of the transgene include PCR, Southern analysis, andWestern analysis.

Other Methods of Corn Transformation

In a further example, friable embryogenic callus derived from immatureembryos of Al88×B73 corn is initiated on a solid medium and transformedbiolistically. Suitable methods are described, for example, in WO98/44140 and U.S. Pat. No. 5,550,318. DNA is provided as a circularplasmid DNA or, alternatively is restricted to provide a linearEPSPS-expression cassette-containing fragment and used followingpurification by agarose gel electrophoresis and electroelution. In afurther example, maize lines including, for example, hybrid lines havingthe genotype A188×B73 are prepared as cell suspensions and transformedby contacting the cell with silicon carbide whiskers coated with DNAusing methods essentially as described by Frame et al (1994, Plant J. 6,941-948).

EXAMPLE 15 In Vitro Measurements of Avena HPPD

In accord with the methods described in the previous examples, AvenaHPPD is found to have a Km value for HPP of˜2.5 μM and a kcat/Km valueof˜2+/−0.6/s/μmol.

At 25° C. and in the absence of glycerol, the rate constants governingdissociation of the complexes with I, II, IV and V are similar to thoseobserved with wheat enzyme and are estimated as>—8×10⁻⁴/s, ˜4×10 ⁴/s,˜2.5×10⁻⁵/s and <4×10⁻⁶/s. Corresponding Ki values were estimatedas>11500 pM, 11400 pM, 710 pM and<30 pM.

Whilst the invention has been particularly described by reference to theintroduction of the Avena gene into soybean, maize and tobacco, theskilled man will recognise that many variations to that specificallydescribed are possible without departing from the scope of the inventionwhich is defined by the appended claims. For example, any suitable planttransformation technique, such as micro-injection, particle mediatedbombardment, polyethylene glycol mediated protoplast transformation,electropdration, protoplast or plant cell sonication etc. may be used tointroduce the polynucleotide or vector of the invention into anymonocot. or dicot. plant material, which may then be regenerated byknown techniques. In particular, for generating plants which areresistant to syncarpic acids (Formula 4) the HPPD encoding sequence fromShewenella Colwelliana is particularly preferred

1. A method of selecting a polynucleotide which encodes a syncarpic acidspecific HPPD inhibitor resistant HPPD enzyme comprising screening apopulation of HPPD enzyme encoding sequences and selecting as thosewhich encode a resistant HPPD enzyme those sequences which encode anenzyme which in comparison with a control HPPD enzyme is at least 2.5fold more resistant to a Formula 4 herbicide compared to a Formula 1herbicide and wherein said control enzyme is selected so as to exhibitsubstantially the same selection of polynucleotides as is obtained whenthe control enzyme is derived from Arabidopsis.
 2. Plant cells whichhave been transformed with a polynucleotide sequence which encodes anHPPD inhibitor resistant HPPD enzyme, wherein the HPPD encoding sequenceis selectable according to claim 1 and/or is derived from an organismselected from the group consisting of Shewenella Colwellina, Vibriovulnificus, Steptomyces avermitilis and Coccidiodes immitus.
 3. Theplant cells according to claim 2, wherein when the cells are dicot cellsthe promoter region used to control expression of the HPPD encodingsequence is derived from the small sub-unit of rubisco, and wherein whenthe cells are monocot cells the promoter region is derived from themaize poly-ubiquitin gene.
 4. The method of claim 1, wherein theresistant HPPD enzyme encoding sequences encode an enzyme which incomparison with a control HPPD enzyme is at least 4 fold more resistantto a Formula 4 herbicide compared to a Formula 1 herbicide and whereinsaid control enzyme is selected so as to exhibit substantially the sameselection of polynucleotides as is obtained when the control enzyme isderived from Arabidopsis.
 5. The method according to claim 1, whereinthe control HPPD is derived from a dicot.
 6. The method according toclaim 5, wherein said dicot is Arabidopsis or tobacco.
 7. The methodaccording to claim 1, wherein the resistance of HPPD enzymes toherbicides is determined by measuring the rate of dissociation of theenzyme/herbicide complex.
 8. The method according to claim 1 wherein theHPPD enzyme encoded by the selected polynucleotide has a kcat/Kmhydroxyphenylpyruvate value in the range from 0.1 to 5 s⁻¹ μM⁻¹ at pH7.0 and 25° C.